Method utilizing purified GP IIb-IIIa to detect the presence of platelet aggregation inhibitors

ABSTRACT

An assay for screening snake venom for the presence or absence of platelet aggregation inhibitors (PAIs) based on specific receptor binding is described. Using this assay, the identification and characterization of PAIs in a wide range of snake venom samples was accomplished. The isolated and purified PAI from several of these active snake venoms is described and characterized. In addition, PAIs lacking the Arg-Gly-Asp (RGD) adhesion sequence but containing K*-(G/Sar)-D wherein K* is a modified lysyl residue of the formula 
     R 1   2  N(CH 2 ) 4  CHNHCO-- 
     wherein each R 1  is independently H, alkyl(1-6C) or at most one R 1  is R 2  --C═NR 3  wherein R 2  is H, alkyl(1-6C) or is NR 4   2  in which each R 4  is independently H or alkyl(1-6C) and R 3  is H, alkyl(1-6C), phenyl or benzyl, or R 2  --C═NR 3  is a radical selected from the group consisting of: ##STR1## where m is an integer of 2-3, and each R 5  is independently H or alkyl(1-6C); 
     and wherein one or two (CH 2 ) may be replaced by O or S provided said O or S is not adjacent to another heteroatom 
     are prepared and shown to specifically inhibit the binding of fibrinogen or yon Willebrand Factor to GP IIb-IIIa.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.08/067,139, filed May 24, 1993 U.S. Pat. No. 5,496,724, which is aContinuation of U.S. application Ser. No. 07/483,229, filed Feb. 20,1990 U.S. Pat. No. 5,318,899, which is a continuation-in-part of U.S.patent application Ser. No. 07/418,028 filed 6 Oct. 1989, now abandoned,which is a continuation-in-part of U.S. patent application Ser. No.07/367,509, filed 16 Jun. 1989, now abandoned.

TECHNICAL FIELD

This invention relates to a group of peptides which are, or are relatedto, platelet aggregation inhibitors isolated and purified from varioussnake venoms. These peptides are useful as therapeutic agents for thetreatment of, and prevention of, platelet-associated ischemic disorders.More specifically, the invention concerns peptides which block specificreceptors for adhesive proteins involved in platelet adherence andaggregation. Furthermore, this invention describes methods for detectingand purifying said polypeptides to substantial homogeneity from snakevenoms, as well as processes for using the primary amino acid sequencesof these polypeptides to prepare active peptides both synthetically andthrough use of recombinant DNA methods.

BACKGROUND ART

Heart disease is the primary cause of death in most western societies.Death from heart disease is often induced by platelet-dependent ischemicsyndromes which are initiated by atherosclerosis and arteriosclerosisand include, but are not limited to, acute myocardial infarction,chronic unstable angina, transient ischemic attacks and strokes,peripheral vascular disease, arterial thrombosis, preeclampsia,embolism, restenosis and/or thrombosis following angioplasty, carotidendarterectomy, anastomosis of vascular grafts, and chroniccardiovascular devices (e.g., in-dwelling catheters or shunts"extracorporeal circulating devices"). These syndromes represent avariety of stenotic and occlusive vascular disorders thought to beinitiated by platelet activation either on vessel walls or within thelumen by blood-borne mediators but are manifested by platelet aggregateswhich form thrombi that restrict blood flow.

Numerous studies have contributed to an understanding of the mechanismof platelet aggregation and thrombus formation. Platelets respond to avariety of blood vessel injuries, such as narrowing of the lumen, plaqueformation, and the presence of foreign bodies (e.g., catheters) and thelike. The response of platelets to these injuries is a sequence ofevents including platelet adherence and activation, and the release ofplatelet granular components, including potent cellular mitogenicfactors. The activated platelet aggregates induce the formation offibrin, which further stabilizes the thrombus.

Much is now known about mechanisms regulating these responses. Althoughunstimulated platelets contain receptors for several adhesive proteinsincluding laminin (VLA 2, VLA 6) and collagen (VLA 2, GPIV, others), theinitial attachment of platelets to subendothelium is believed to bemediated by the binding of platelet membrane glycoprotein (GP) Ib to theimmobilized yon willebrand factor. Subsequent platelet activation can beinitiated by one or more of the known physiological agonists including:ADP, epinephrine, thrombin, collagen, and thromboxane A2.

Platelet aggregation is mediated by GP IIb-IIIa complex on the plateletmembrane surface. GP IIb-IIIa exists on the surface of unstimulatedplatelets in an in-active form. When platelets are activated by adhesionand the physiological agonists, the GP IIb-IIIa also becomes activatedsuch that it becomes a receptor for fibrinogen (Fg), von WillebrandFactor (vWF), and fibronectin (Fn) (see Phillips et al., Blood (1988)71:831-843); however, it is the binding of fibrinogen and/or vonWillebrand factor that is believed to be principally responsible forplatelet aggregation and thrombus formation in vivo. Therefore,substances which specifically inhibit the binding of fibrinogen or vonWillebrand factor to GP IIb-IIIa inhibit platelet aggregation and couldbe candidates for inhibiting thrombus formation in vivo.

Platelet GP IIb-IIIa is now known to be a member of a superfamily ofstructurally related adhesive protein receptors known collectively asthe "integrins." Like GP IIb-IIIa, all integrins known to date are twosubunit molecules with a larger alpha-subunit (e.g., GP IIb) and asmaller beta-subunit (e.g., GP IIIa). There is a high degree of homologybetween the known sequences of the integrin subunits indicating that theintegrins evolved from a common precursor. Integrins function in avariety of cellular adhesions and have been found in leucocytes,endothelial cells, smooth muscle cells and other cells in thevasculature. Because integrins are widely distributed, while GP IIb-IIIais restricted to platelets, a preferred antiaggregating agent wouldselectively inhibit GP IIb-IIIa as opposed to other integrins.

Several classes of peptides have been disclosed which block the bindingof adhesive proteins to activated platelets and inhibit plateletaggregation (see Hawiger et al., U.S. Pat. No. 4,661,471; and Rouslahtiet al., U.S. Pat. Nos. 4,614,517; 4,578,079; 4,792,525; and UKapplication GB 2,207,922A). In one class of peptides, the sequence RGDis critical, and the tetrapeptide sequences RGDS, RGDT, RGDC, have beenused specifically. The amino acid sequence RGDX is found in a variety ofadhesive proteins including Fg, Vn, vWF and Fn. This sequence has beendemonstrated to play an important role in the interaction of adhesiveproteins with adhesive protein receptors because peptides containingthis sequence block the binding of adhesive proteins. See, e.g.,Pierschbacher, M. D., et al., J Biol Chem (1987) 262:17294-17298;Ruggeri et al., Proc Natl Acad Sci (USA) (1986) 83:5708-5712; andRouslahti et al., Cell (1986) 44:517-518.

The structural variations permitted in RGD-containing peptides have beenexplored by Pierschbacher, M. D. et al. J Biol Chem (supra). In thesestudies, it was found that manipulating the RGD-containing sequence notonly effected the activity related to inhibition of binding offibronectin or vitronectrin to substrate, but could also effectdifferentiation between binding of the two ligands. The peptide sequenceGRGDSPC which was taken from the cell attachment domain of fibronectinwas used as a model peptide. Certain substitutions, such as replacementof L-Arg with D-Arg seem to have no effect on the binding of eitherligand, but substituting D-Ala for Gly or D-Asp for L-Asp destroyed theinhibition activity. While substituting D-Ser for L-Ser reducedinhibition of vitronectin interaction with vitronectin receptor, therewas little effect on fibronectin interaction with fibronectin receptor;substitution of Ash for Ser resulted in a peptide that had enhancedinhibition of fibronectin binding, and a decreased effect on vitronectinbinding. Alternate substitutions for Ser had other effects. Threoninesubstituted for Set gave a peptide with increased inhibition of bindingto the vitronectin receptor; substitution of L-Pro led to an inactivepeptide. A cyclic peptide was also prepared of the sequenceGly-Pen-Gly-Arg-Gly-Asp-Ser-Pro-Cys-Ala, wherein "Pen" is penicillamineand a disulfide bridge was formed between the Pen and Cys. In the viewof the authors, penicillamine had the function of increasingconformational restraints on the ring whereas the N-terminal Gly andcarboxy-terminal Ala were added to distance the free amino and carboxylgroups from the ring. This cyclic peptide was able to inhibitvitronectin binding more strongly than the same peptide beforecyclization, but was ineffective in inhibiting fibronectin binding.

Recently, an antithrombotic peptide with a modification of the RGDsequence having the "R" residue alkylated was reported by Samanen, J.,et al., J Cell Biochem (1990) Suppl 14A:A229.

A separate class of inhibitory peptides utilizes peptide sequencesmodeled on the carboxyl terminal sequence derived from the gamma chainof fibrinogen, the dodecapeptide HHLGGAQKAGDV (Kloczewiak et al.,Biochemistry (1989) 28:2915-2919; Timmons et al., (Ibid), 2919-2923 U.S.Pat. No. 4,661,471 (supra); EP application 298,820,). Although thissequence inhibits Fg and vWF binding to GP IIb-IIIa and subsequentplatelet aggregation, the usefulness of this peptide is limited becauseit has a low affinity of interaction with platelet receptors (IC₅₀=10-100 uM).

Recently, several groups have isolated and characterized a new class oflow molecular weight polypeptide factors from snake venoms which haveextremely high affinity for the GP IIb-IIIa complex. Huang, T.-F., etal., J Biol Chem (1987) 262:16157-16163; Huang, T.-F., et al.,Biochemistry (1989) 28:661-666 report the primary structure oftrigramin, a 72 amino acid peptide containing RGD and 6 disulfidebridges isolated from Trimeresurus gramineus. Gan, Z.-R., et al., J BiolChem (1988) 263:19827-19832, report the properties and structure ofechistatin, a 49 amino acid peptide also containing RGD and 4 putativedisulfide bridges which is isolated from Echis carinatus. Williams, J.A., et al., FASEB Journal (1989) 3:A310, Abstr. No. 487m, report thesequence and properties of the related peptides elegantin, albolabrin,and flavoviridin.

Included in this group of inhibitory peptides from snake venoms arealboabrin isolated from Trimeresurus albolabris, elegantin isolated fromT. elegans, flavoviridin isolated from T. flavoviridis, batroxostatinisolated from Bothrops atrox, bitistatin isolated from Bitis arietansreported by Niewiarowski, S., et al., Thromb haemostas (1989) 62:319(Abstr. SY-XIV-5). In addition, applaggin has been purified fromAgkistrodon p. piscivorus and reported by Chao, B., et al., ThrombHaemostas (1989) 62:50 (Abstr. 120) and halysin, purified fromAgkistrodon halys which was reported by Huang, T. F., et al., Thrombhaemostas (1989) 62:48 (Abstr. 112). All of these peptides show a highdegree of sequence homology. In addition, all of the peptides reportedto date from snake venoms which inhibit the binding of adhesive proteinsto integrin receptors contain the RGD sequence.

Although these reported snake venom factors are potent plateletaggregation inhibitors in vitro, these peptides also bind with highaffinity to other members of the adhesive protein receptors such as thevitronectin and fibronectin receptors (Knudsen, K. A., et al., Exp CellRes (1988) 179:42-49; Rucinski, B., et. al., Thromb Haemostas (1989)62:50 (Abstr. 120). This lack of specificity of snake venom factors forGP IIb-IIIa is an undesirable feature of their therapeutic use asinhibitors of thrombus formation, because they have the potential ofaffecting the adhesive properties of other cells in the vasculature,particularly those adhesions mediated by integrins.

Another approach developed for the generation of platelet thrombusinhibitors has been the use of murine anti-GP IIb-IIIa monoclonalantibodies which block the binding of the adhesive proteins tostimulated platelets. These monoclonal antibodies have been used toprevent coronary artery reocclusion after reperfusion with tissueplasminogen activator in dogs (Yasuda, T., et al., J Clin Invest (1988)81:1284-1291) and to prevent cyclic reduction of flow in injured caninecoronary arteries with a high grade stenosis. Potential side effects ofthe use of such monoclonal antibodies in humans may result from theirlong-lasting effects and from their potential immunogenicity.

Clearly, additional therapeutic treatment regimens are needed forpreventing or at least mitigating undesirable thrombus formation. Inparticular, therapeutic agents capable of blocking or inhibitingthrombus formation at specific locations without compromising hemostasisand without affecting other cellular adhesions, would provide majortherapeutic benefits. Ideally, these agents should be potent, specificfor GP IIb-IIIa, and nonimmunogenic to most patients; they also shouldbe easy to administer, stable and economical to produce. Further, theseagents should act transiently and be capable of functioning at theearliest stages of thrombus formation, without interfering withlong-term hemostasis. The present invention fills these and otherrelated needs.

DISCLOSURE OF THE INVENTION

The invention provides a simple screening procedure to identify lowmolecular weight (<10kd) factors in snake venom or other biologicalsources that specifically inhibit thrombus formation mediated byplatelet aggregation. This procedure takes advantage of theunderstanding that platelet aggregation is primarily effected throughbinding of fibrinogen and/or vWF to GP IIb-IIIa at the surface ofplatelets when the platelets are treated with appropriate stimuli, suchas ADP. By using these criteria, i.e., inhibition of binding offibrinogen and/or vWF to isolated receptor and analogous criteriarelated to inhibition of binding of fibronectin (Fn) to fibronectinreceptor (Fn/FnR binding) and vitronectin to vitronectin receptor(Vn/VnR binding), as well as the binding of other factors, such as Fnand Vn to GP IIb-IIIa, a specificity profile for the plateletaggregation inhibitor (PAI) can be rapidly and conveniently obtained.This approach has been used to screen and characterize an extensivepanel of snake venoms for the presence or absence of PAI, tocharacterize the specificity of PAI identified from this panel for theirspecificity at inhibiting binding to GP IIb-IIIa as opposed toinhibiting other integrins, and to identify active peptides which arederivatives of these PAIs.

Accordingly, in one aspect, the invention is directed to a rapidscreening method for the presence or absence of PAI in a biologicalfluid, which method comprises contacting the fluid with isolated GPIIb-IIIa in a test reaction in the presence of fibrinogen and comparingthe amount of fibrinogen bound to GP IIb-IIIa in this test reaction withthe amount of fibrinogen bound to GP IIb-IIIa in a control reaction. Themethod may further include test and control reactions which involvecontacting Fn with Fn receptor, Vn with Vn receptor, Fn with GPIIb-IIIa, or vWF with GP IIb-IIIa to characterize the specificity of thePAI.

In another aspect, the invention is directed to novel PAI in isolatedform which is identified in, and can be isolated from, active snakevenom according to the methods of the invention. In particular, theinvention relates to PAI, in isolated form, which can be isolated fromEchis colorata, Eristicophis macmahonii; A. hypnale, A. acutus, A.piscivorous leucostoma, A. piscivorus conanti; Bothrops asper; Bothropscotiara, B. jararaca, B. jararacussu, B. lansbergi, B. medusa, B.nasuta, B. neuwiedi, B. pradoi, B. schlegli; Crotalus atrox, C.basilicus, C. cerastes cerastes, C. durissus durissus, C. durissustotonatacus, C. horridus horridus, C. molossus molossus, C. ruber ruber,C. viridis cereberus, Crotalus v. helleri, Crotalus v. lutosus, Crotalusv. oreganus, Crotalus v. viridis; Lachesis mutas; Sistrurus catenatustergeminus, and Sistrurus milarus barbouri.

Preferred are PAIs in isolated form prepared from, or having the aminoacid sequences of, those obtained from Eristicophis macmahonii(eristicophin); Bothrops cotiara (cotiarin); B. jararacussu; Crotalusatrox (crotatroxin); Crotalus basilicus (basilicin); C. cerastescerastes (cerastin); C. durissus totanatacus; C. durissus durissus; C.h. horridus (horridin); Crotalus m. molossus (molossin); C. ruber ruber(ruberin); Crotalus viridis lutosus (lutosin); C. v. viridis (viridin);Crotalus v. oreganus; Crotalus v. helleri; Lachesis mutas (lachesin);Sistrurus catenatus tergeminus (tergeminin); and S. milarus barbouri(barbourin).

Especially preferred are eristicophin, cotiarin, crotatroxin, cerastin,horridin, ruberin, lachesin, basilicin, lutosin, molossin, viridin,tergeminin and barbourin.

The invention also includes peptides of the amino acid sequences asdescribed above which are truncated and/or modified forms of thenaturally occurring peptides and/or have one or more peptide linkagesreplaced by alternate linkages such as --CH₂ NH-- or --CH₂ CH₂ --.

In a preferred aspect, the invention relates to PAI in isolated formwhich can be prepared from active snake venom identified by the methodof the invention, and shown to specifically inhibit the bindingfibrinogen (Fg) and/or von Willebrand Factor (vWF) to GP IIb-IIIa, andtheir truncated and/or modified forms.

In still another preferred aspect, the invention relates to PAI of snakevenom in isolated form wherein the sequence responsible for binding tothe adhesive protein receptor includes the sequence KGD.

In another major aspect, the invention is directed to a group ofpeptides or peptide-related compounds in general which are plateletaggregation inhibitors that are capable of inhibiting binding of Fg orvWF to GP IIb-IIIa at a substantially higher potency than that at whichthey inhibit binding of vitronectin to vitronectin receptor orfibronectin to fibronectin receptor. These peptides are characterized byhaving the binding sequence K*GDX in place of the RGDX binding sequencewhich is found in the prior art PAI proteins. K* is a substituted orunsubstituted lysyl residue of the formula R¹ ₂ N(CH₂)₄ CHNHCO-- whereineach R¹ is independently H or a substituent which is sufficientlyelectron donating so as not to destroy the bacicity of the adjacentnitrogen, and wherein one or two of the methylene residues mayoptionally be substituted by O or S, as described below. The barbourinPAI isolated from S. milarus barbouri is one illustration of this seriesof peptides. However, shorter forms of this peptide can also be used, aswell as analogous sequences which also contain 1-10 amino acid residuemodifications elsewhere in the peptide chain, and/or replacement ofpeptide linkages with alternate linkages. Other illustrative embodimentsinclude isolated PAI peptides having a native RGDX sequence wherein thisis replaced by K*GDX. As in the case of barbourin, these isolated PAImay be otherwise in native form, or may be truncated and/or may contain1-10 amino acid residue substitutions or deletions, and/or may havenon-peptide linkages substituted for peptide linkages.

Another group of compounds which falls within the scope of the inventionis that wherein the foregoing compounds are as described, except thatthe glycyl residue in the RGD or K*GD sequence is replaced by a sarcosylresidue. This class of compounds retains the potency and specificity ofthe related RGD or K*GD-containing peptides.

Another illustrative group of embodiments are peptides or modifiedpeptides having specific PAI activity of the formula ##STR2## wherein K*is a substituted or unsubstituted lysyl residue of the formula R¹ ₂N(CH₂)₄ CHNHCO-- as described above,

wherein each R¹ is independently H, alkyl (1-6C), or at most one R¹ isR² --C═NR³ wherein R² is H, alkyl(1-6C) or is NR⁴ ₂ in which each R⁴ isindependently H

or alkyl(1-6C) and R³ is H, alkyl(1-6C), phenyl or benzyl, or R² --C═NR³is a radical selected from the group consisting of: ##STR3## where m isan integer of 2-3, and each R⁵ is independently H or alkyl(1-6C);

and wherein one or two (CH₂) may be replaced by O or S provided said Oor S is not adjacent to another heteroatom;

AA₁ is a small, neutral (polar or nonpolar) amino acid and n1 is aninteger of 0-3;

AA₂ is a neutral, nonpolar large (aromatic or nonaromatic) or a polararomatic amino acid and n₂ is an integer of 0-3;

AA₃ is a proline residue or a modified proline residue (as definedbelow) and n₃ is an integer of 0-1;

AA₄ is a neutral, small amino acid and n4 is an integer of 0-3;

each of X₁ and X₂ is independently a residue capable of forming a bondbetween X₁ and X₂ to obtain a cyclic compound as shown; and

each of Y₁ and Y₂ is independently a noninterfering substituent or maybe absent;

wherein one or more peptide linkages may optionally be replaced by alinkage selected from the group consisting of --CH₂ NH--, --CH₂ S--, CH₂CH₂ --, --CH═CH-- (cis and trans), --COCH₂ --, --CH(OH)CH₂ -- and --CH₂SO--.

Other aspects of the invention are concerned with recombinant methodsand materials related to the synthesis of these and other relatedpeptides, to methods of in vitro synthesis thereof, to pharmaceuticalcompositions containing these compounds, and to methods to inhibitplatelet aggregation and thrombus formation using these compounds andcompositions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows inhibition of the binding of fibrinogen to GP IIb-IIIa bypartially purified snake venoms.

FIGS. 2A, 2B, and 2C show the dose-response adhesion inhibition ofCentricon-10 ultrafiltrates of crude venoms in both fibrinogen/GPIIb-IIIa and vitronectin/vitronectin receptor assays. Selectivity ofsnake venom peptides in GPIIb-IIIa and vitronectin receptor bindingassays is shown. Sistrurus m. barbouri venom is used in FIG. 2A.Crotalus ruber venom is used in FIG. 2B. Crotalus basilicus venom isused in FIG. 2C .

FIG. 3 shows the HPLC profile of crude PAI from Eristicophis macmahonivenom. The cross hatched area contains the biologically activefractions.

FIG. 4 shows the slow gradient HPLC profile of PAI fractions from FIG.3. The cross-hatched area contains the bioactive fractions.

FIG. 5 shows the analytical HPLC profile of PAI fractions from FIG. 4 todemonstrate purified PAI from Eristicophis macmahoni venom.

FIGS. 6-1 to 6-3 show the complete amino acid sequences of eristicophin,barbourin, tergeminin, lachesin, ruberin, cerastin, cotiarin,crotatroxin, and horridin and enzyme digestion fragments determined byautomated Edman degradation.

FIG. 7 depicts the HPLC profile of PAI obtained from G-50 fractions ofcrude Sistrurus c. tergeminus venom. The cross-hatched area contains thebioactive fractions.

FIG. 8 depicts the HPLC profile of PAI fractions from FIG. 7 to showpurified PAI from Sistrurus catenatus tergeminus venom.

FIG. 9 shows the activity of the purified tergeminin PAI of FIG. 8 ininhibiting binding in several receptor assays.

FIG. 10 depicts the HPLC profile of platelet aggregation inhibitorobtained from G-50 fractions of crude Sistrurus m. barbouri venom. Thecross-hatched areas contain the bioactive fractions.

FIG. 11 depicts the HPLC profile of active PAI fractions of FIG. 10 toshow purified PAI from Sistrurus milarus barbouri.

FIGS. 12-1 and 12-2 compare the amino acid sequences of a number ofsnake venom PAIs to that of barbourin.

FIG. 13 depicts the HPLC profile of crude PAI from Lachesis mutas venom.Cross-hatched areas contain the biologically active fractions.

FIG. 14 depicts the slow gradient HPLC profile of the PAI activefractions from FIG. 13. Cross-hatched area contains the biologicallyactive fractions.

FIG. 15 depicts the analytical HPLC profile of PAI fractions of FIG. 14from Lachesis mutas to show purified PAI from Lachesis mutas venom.

FIG. 16 depicts the HPLC profile of crude PAI from Crotalus viridisviridis venom. Cross-hatched area contains the biologically activefractions.

FIG. 17 depicts the HPLC profile of the PAI fractions of FIG. 16 to showpurified PAI from Crotalus viridis viridis venom.

FIG. 18 shows the dose-response effects of purified snake venom peptidesto inhibit fibrinogen/GP IIb-IIIa binding as compared to echistatin.

FIGS. 19A, 19B and 19C show the dose-response effects of purified snakevenom peptides to inhibit ADP (4 uM) induced human platelet aggregationin platelet rich plasma (PRP), as compared to echistatin.) FIG. 19Ashows the effect of Sistrurus m. barbouri PAI barbourin. FIG. 19B showsthe effect of Eristicophus macmahoni PAI eristicophin. FIG. 19C showsthe effect of Echis carnitus PAI echistatin.

FIG. 20 shows the activity profile from HPLC fractionation of C. c.cerastes venom. Cross-hatched area contains the biologically activefractions.

FIG. 21 shows the results of HPLC analysis of the active fractions ofFIG. 20 to show purified PAI from Crotalus cerastes cerastes venom.

FIG. 22 shows the activity profile of HPLC fractionation of PAI from C.ruber ruber. Cross-hatched area contains biologically active fractions.

FIG. 23 shows the activity profile of an analytical C-18 column onhomogeneous peptide obtained from C. atrox.

FIG. 24 shows the analytical HPLC profile of the homogeneous peptideisolated from Bothrops cotiara.

FIG. 25 shows the dose-response effects of purified cotiarin oninhibiting the binding of fibrinogen to GP IIb-IIIa and inhibition ofthe binding of vitronectin to the vitronectin receptor.

FIG. 26 shows the effects of purified snake venom peptides on binding offibrinogen to GP IIb-IIIa and vitronectin to the vitronectin receptor.The values indicate the concentration of purified peptide which inhibitsthe binding of fibrinogen to GPIIb-IIIa or vitronectin to thevitronectin receptor by 50% (IC50). The symbol ">" indicates that theIC50 is greater than this value, which is the highest concentrationexamined to date. Note that substitution of the arginine in the "RGDW"sequence in Eristicophin with a lysine (KGDW) imparts specificity forinhibiting fibrinogen binding to GPIIb-IIIa versus vitronectin to thevitronectin receptor.

FIG. 27 shows the results of binding activity for analog #1, E²⁸ L⁴¹ C⁶⁴!barbourin(28-73), with regard to GP IIb-IIIa and vitronectin receptor.

FIG. 28 shows the ability of synthetic eristicophin analog K²⁹!-ensticophin (4-51) to inhibit the binding of fibrinogen to GP IIb-IIIaand inability to inhibit the binding of virtonectin to the vitroncetinreceptor.

FIGS. 29A and 29B show the ability of linear and cyclic RGDW compoundsand linear and cyclic KGDW compounds to inhibit the binding offibrinogen to GP IIb-IIIa.

FIGS. 30A, 30B, 30C, 30D and 30E show the ability of various KGDWanalogs to inhibit binding of fibrinogen to GP IIb-IIIa and inhibitbinding of vitronectin to vitronectin receptor. FIG. 30A representsAnalog #7. FIG. 30B represents Analog #8. FIG. 30C represents Analog #4.FIG. 30D represents Analog #5. FIG. 30E represents Analog #6.

FIG. 31 shows the ability of various native and synthetic plateletaggregation inhibitors to inhibit the attachment of M21 melanoma cellsto vitronectin.

FIG. 32 shows the ability of RGDS and a cyclic RGD compound to inhibitthe attachment of M21 melanoma cells to vitronectin and the lack ofability of a cyclic KGDW analog to inhibit the attachment of M21melanoma cells to vitronectin.

FIG. 33 shows the activity of analog number 60, Mpr-(Har)-G-D-W-P-C-NH₂,in inhibiting aggregation of platelets and cell adhesion to vitronectin.

FIG. 34 shows the activities of FIG. 33 for analog 19,Mpr-K-G-D-W-P-C-NH₂.

FIG. 35 shows the initiation of cyclic flow reductions (CFRs) in an openchest dog model of thrombosis (Folts Model).

FIG. 36 shows the effects of a 10 mg bolus dose administration on theCFRs initiated in the open chest dog model of thrombosis (Folts Model).

FIG. 37 shows the effects of a 40 mg bolus dose administration on theCFRs initiated in the open chest dog model of thrombosis (Folts Model).

FIG. 38 shows the full-length DNA sequence encoding the amino acidsequence of barbourin(1-73).

FIG. 39 shows the DNA sequence encoding M⁻¹, L⁴¹ !barbourin(1-73)ligated to a PhoA leader sequence.

FIG. 40 shows the DNA sequence encoding analog #1, E²⁸, L⁴¹, C⁶⁴ !barbourin (28-73), linked to a PhoA leader sequence for expression inbacteria.

FIGS. 41A and 41B show oligonucleotides utilized in a PCR reaction toobtain DNA encoding analog #1. The amino acids included in the analogper se are shown in boldface type.

FIG. 42 shows the junction sequence of tandem repeats of the analog#1-encoding DNA.

FIGS. 43A and 43B show a diagram of the truncated barbourin gene astandem repeats.

MODES OF CARRYING OUT THE INVENTION

The invention provides platelet aggregation inhibitors (PAI) which maybe isolated from snake venom which has been identified as active by theassay methods of the invention and compounds which have similarstructures and are synthesized using standard in vitro techniques, suchas solid phase peptide synthesis, or using recombinant methods, orcombinations of these. Some of these inhibitors are uniquely specificfor inhibition of platelet aggregation and do not inhibit alternatebinding within the integrin family. Others have different ranges ofspecificity. The sections below describe the isolation of naturallyoccurring PAI from snake venom; the design of inhibitors which are ofsubstantially higher potency in inhibiting platelet aggregation than ininhibiting, for example, vitronectin/vitronectin receptor interaction byincorporating a K*GD sequence in preference to RGD; methods ofsynthesizing these peptides; methods of recombinant production;antibodies raised against the invention peptides; the assay method whichpermits identification of snake venoms which contain PAI; and theadministration and utility of the PAI of the invention.

By PAI is meant a factor which is capable of preventing the aggregationof stimulated platelets in standard assays, for example those describedby Gan, Z. -R., et al., and Huang, T.-F., et al., (supra). In theseassays, washed platelets are combined with fibrinogen, Ca⁺² and thematerial to be tested. The platelets are stimulated with ADP (or otherknown stimulators or combinations thereof) and aggregation (or lackthereof) is observed using, for example, a commercially availableaggregometer.

Some of the PAIs of the invention are identified as specific for theinhibition of binding of fibrinogen and/or vWF to GP IIb-IIIa. It isunderstood that specificity is a matter of degree; therefore, a PAI"specific for inhibition of Fg or vWF binding to GP IIb-IIIa inhibitsthis binding substantially more than it inhibits the binding of Fn toFnR, or Vn to VnR. By "substantially more" is meant that either the %inhibition is at least twofold greater at a given concentration of PAIor that the concentration of PAI that causes 50% inhibition is at leasttwofold less for Fg or vWF/GP IIb-IIIa binding inhibition than foralternate ligand/receptor binding.

Isolated Native PAI and Purification Methods

The platelet aggregation inhibitors (PAI) of the invention include lowmolecular weight peptides which can be prepared in isolated form, asdescribed below, from snake venom which has been identified as "active",i.e., has been found to contain PAI using the method of the invention,which is described hereinbelow.

The invention method permits ready identification and characterizationof the presence of an effective PAI in snake venom which selectivelyinhibits binding to GP IIb-IIIa as opposed to other integrins as, forexample, the vitronectin receptor and the fibronectin receptor. Uponsuch identification, and, optionally and optimally, characterization,the PAI can be isolated and purified using a variety of standardtechniques illustrated herein and disclosed in the art. For example, acombination of separation based on molecular weight (typically recoveryof substances of <10kd), ion exchange chromatography, and reverse phaseHPLC can be used. Other techniques can also be employed, but a workableprocedure applicable to PAI from any active snake venom is as follows:

About 10-1000 mg venom is dissolved in dilute acetic acid and applied toa sizing column, such as Sephadex G-50, and eluted in the same solvent.Fractions are assayed for activity using the Fg/GP IIb-IIIa bindingassay of the invention, a standard platelet aggregation assay (PAA) orany similar assay relying on the adhesive protein binding activity of GPIIb-IIIa. Alternatively, the <10kd fraction of the fraction of the venomcan be recovered using ultrafiltration and similarly assayed.

The low MW fraction isolated by either procedure is then loaded onto apreparative C-18 HPLC column, such as a C-18 Delta Pak reverse phaseHPLC column, available from Waters, preequilibrated in 0.1%trifluoroacetic acid (TFA)/8% acetonitrile. The adsorbed PAI is theneluted using a gradient of 8%-60% acetonitrile in 0.1% TFA. The slope ofthe gradient and flow rate are optimized using routine procedures.Active fractions are determined by PAA or by the invention receptorbinding method. The active fractions are then pooled, concentrated, andtested for homogeneity using analytical HPLC or SDS-PAGE. Furtherreverse-phase HPLC gradient purification is applied until the recoveredPAI is homogenous.

PAIs of the invention, obtainable by the foregoing or other purificationmethods include those from venoms selected from the group consisting ofEchis colorata, Eristicophis macmahonii; A. hypnale, A. acutus, A.piscivorous leucostoma, A. piscivorus conanti; Bothrops asper; Bothropscotiara, B. jararaca, B. jararacussu, B. lansbergi, B. medusa, B.nasuta, B. neuwiedi, B. pradoi, B. schlegli; Crotalus atrox, C.basilicus, C. cerastes cerastes, C. durissus durissus, C. durissustotonatacus, C. horridus horridus, C. molossus molossus, C. ruber ruber,C. viridis cereberus, Crotalus v. helleri, Crotalus v. lutosus, Crotalusv. oreganus, Crotalus v. viridis; Lachesis mutas; Sistrurus catenatustergeminus, and Sistrurus milarus barbouri.

Preferred are PAIs in isolated form prepared from, or having the aminoacid sequences of, those obtained from Eristicophis macmahonii(eristicophin); Bothrops cotiara (cotiarin); B. jararacussu; Crotalusatrox (crotatroxin); Crotalus basilicus (basilicin); C. cerastescerastes (cerastin); C. durissus totonatacus; Crotalus d. durissus; C.h. horridus (horridin); Crotalus m. molossus (molossin); C. ruber ruber(ruberin); Crotalus viridis lutosus (lutosin); C. v. viridis (viridin);Crotalus v. oreganus; Crotalus v. helleri; Lachesis mutas (lachesin);Sistrurus catenatus tergeminus (tergeminin); and S. milarus barbouri(barbourin). Particularly preferred are PAI specific for inhibiting Fgor vWF/GP IIb-IIIa binding, e.g., that from Sistrurus m. barbouri.

Especially preferred are eristicophin, cotiarin, crotatroxin, cerastin,horridin, ruberin, lachesin, basilicin, lutosin, molossin, viridin,tergeminin and barbourin.

The purified PAI of the invention can be sequenced using standardprocedures, thus permitting synthesis using standard solid phasetechniques (in particular for shorter forms of the PAI) or recombinantproduction. For example, an Applied Biosystems Sequenator can be usedfollowing carboxyamido methylation or pyridylethylation of the peptideas described by Huang et al., J Biol Chem (1987) 262:16157-16163followed by desalting of the sample on a C-18 Delta Pak column using0.1% TFA and acetonitrile.

It is understood that the isolated PAI of determined sequence can, whensynthesized in vitro, be modified by sequence alterations which do notdestroy activity. In general, these modified forms will differ from thenative forms by 1-10, preferably 1-4, amino acid substitutions or willbe truncated forms. In addition, one or more peptide linkages may bereplaced by alternate linkages as described hereinbelow. A particularlypreferred substitution is replacement of RGD by KGD to confer GPIIb-IIIa specificity as described below.

The PAI of Sistrurus m. barbouri has been purified to homogeneity andsequenced, and termed "barbourin" Unlike the adhesive proteins for GPIIb-IIIa so far identified and the peptides from snake venoms that blockGP IIb-IIIa function, barbourin does not contain the standardArg-Gly-Asp sequence of the adhesive proteins known in the art. Theapparent binding sequence in barbourin is Lys-Gly-Asp-(Trp). Thepresence of the KGD sequence in the apparent binding region of thispeptide is especially surprising in view of the observation thatreplacement of Lys for Arg in small synthetic peptides based on the RDGXsequence greatly decreases the ability of these peptides to bind tointegrin receptors (Pierschbacher et al., Proc Natl Acad Sci (USA)(1984) 81:5985-5988; Williams et al., Thromb Res (1987) 46:457-471);Huang et al., J.Biol Chem (1987) 262:16157-16163. It is thought thatthis substitution may in part be responsible for the specificity of thebarbourin peptide to inhibit Fg and vWF binding to GP IIb-IIIa, versus,for example, inhibition of vitronectin binding to the vitronectinreceptor.

K*GDX-Containing Peptides

The "barbourin" peptide isolated by the method of the invention has beenshown to have the binding sequence KGDX in contrast to the RGDX found inthe PAI compounds of the prior art. The presence of the KGDX in this PAIsequence appears to be associated with a preferential affinity for GPIIb-IIIa as opposed to the vitronectin or fibronectin receptors. Theeffect of the substitution of a lysyl residue for an arginine in thesequence appears to be associated with increased length of the sidechainalong with retained basicity of the nitrogen as is further describedhereinbelow. Surprisingly, it appears that it is not the lysyl residueper se which accounts for the enhanced activity and specificity, butrather the spacing provided by this homologous extension of the replacedarginine. Thus, the peptides of the invention which contain K*GDX in thebinding sequence are substantially more potent in inhibiting the bindingof Fg or vWF to GP IIb-IIIa as compared to their ability to inhibit thebinding of vitronectin to the vitronectin receptor and the binding offibronectin to the fibronectin receptor. As stated above, by"substantially more" potent in inhibiting the preferred binding is meantthat the percent inhibition is at least 2-fold greater at a setconcentration of inhibitor or that the concentration of PAI that causes50% inhibition is at least 2-fold less for the binding of Fg or vWF toGP IIb-IIIa than for the binding of alternate ligands to otherintegrins.

As used herein K* refers to a lysyl residue which is unsubstituted, orwhich contains substitutions for the hydrogens on the epsilon aminogroup. The substituents must be sufficiently electron donating so as tomaintain the basicity of the nitrogen to which they are attached. Thus,K* is defined as a lysyl residue of the formula R¹ ₂ N(CH₂)₄ CHNHCO--,

wherein each R¹ is independently H, alkyl (1-6) or at most one R¹ is R²--C═NR³ wherein R² is H, alkyl(1-6C) or is NR⁴ ₂ in which each R⁴ isindependently H or alkyl(1-6C) and R³ is H, alkyl(1-6C), phenyl orbenzyl, or R² --C═NR³ is a radical selected from the group consistingof: ##STR4## where m is an integer of 2-3, and each R⁵ is independentlyH or alkyl(1-6C);

and wherein one or two (CH₂) may be replaced by O or S provided said Oor S is not adjacent to another heteroatom.

"Alkyl" is conventionally defined as a straight or branched chain orcyclic hydrocarbyl residue of the indicated number of carbon atoms suchas methyl, ethyl, isopropyl, N-hexyl, 2-methylbutyl, cyclohexyl and thelike.

As the essential feature of the preferential inhibition of bindingappears to reside in the substitution of K* for R of RGDX, one class ofpeptides or peptide-related compounds of the invention comprisesnaturally occurring platelet aggregation inhibitors which ordinarilycontain RGDX in the binding sequence whereby these forms are modified bysubstituting K* for R in this sequence. Included in the invention arethe native peptides having this substitution, as well as their fragmentsof sufficient length to be effective in selectively inhibiting thebinding of adhesive proteins to GP IIb-IIIa and fragments or full-lengthpeptides which have irrelevant substitutions in positions of the peptidewhich do not destroy this activity. For the most part, the fragmentswill contain residues corresponding to the length of a peptide chain ofat least 7 amino acids if the conformation is controlled by, forexample, cyclization, and are of greater length if there is no suchconformational control. In general, aside from the K*GDX requiredsequence, there may be 1-10, preferably 1-4, and more preferably 1-3amino acid substitutions in the non-KGDX portion of the peptides.

Additionally, the G of RGDX or K*GDX may be replaced by a sarcosineresidue.

In addition, one or more of the peptide bonds may be optionally replacedby substitute linkages such as those obtained by reduction orelimination. Thus, one or more of the --CONH-- peptide linkages can bereplaced with other types of linkages such as --CH₂ NH--, --CH₂ S--, CH₂CH₂ --, --CH═CH-- (cis and trans), --COCH₂ --, --CH(OH)CH₂ -- and --CH₂SO--, by methods known in the art. The following references describepreparation of peptide analogs which include these alternative-linkingmoieties: Spatola, A. F., Vega Data (March 1983), Vol. 1, Issue 3,"Peptide Backbone Modifications" (general review); Spatola, A. F. in"Chemistry and Biochemistry of Amino Acids, Peptides and Proteins," B.Weinstein, eds., Marcel Dekker, New York, p. 267 (1983) (generalreview); Morley, J. S., Trends Pharm Sci (1980) pp. 463-468 (generalreview); Hudson, D. et al. Int J Pept Prot Res (1979) 14:177-185 (--CH₂NH--, CH₂ CH₂ --); Spatola, A. F. et al., Life Sci (1986) 38:1243-1249(--CH₂ --S); Hann, M. M. J Chem Soc Perkin Trans I (1982) 307-314(--CH--CH--, cis and trans); Almquist, R. G., et al., J Med Chem (1980)23:1392-1398 (--COCH₂ --); Jennings-White, C. et al. Tetrahedron Lett(1982) 23:2533 (--COCH₂ --); Szelke, M., et al., European Appln. EP45665 (1982) CA: 97:39405 (1982) (--CH(OH)CH₂ --); Holladay, M. W. etal. Tetrahedron Lett (1983) 24:4401-4404 (--C(OH)CH₂ --); and Hruby, V.J. Life Sci (1982) 31:189-199 (--CH₂ --S--). Particularly preferred is--CH₂ NH--.

Examples of fragments and/or modified forms of the naturally-occurringsnake venom PAI include E²⁸, L⁴¹, C⁶⁴ !barbourin(28-73) of the sequence

1,28 ECADGLCCDQCRFLKKGTVCRVAKGDWNDDTCTGQSCDCPRNGLYG 46,73 and K²⁹!eristicophin(4-51) of the sequence

4,4 EEPCATGPCCRRCKFKRAGKVCRVAKGDWNNDYCTGKSCDCPRNPWNG 51,51

In this notation, the size of the fragment is noted in parentheses afterthe name by the numbers of the amino acids which are included in thefragment, and the bracketed prefix letters and numbers indicate aminoacid substitutions at the numbered positions in the native full-lengthpeptide. Thus, for the barbourin fragment above, the length of thefragment spans residues 28-73 inclusive of the native sequence and theamino acids originally in positions 28, 41 and 64 of the numbered nativesequence have been replaced by Glu (E), Leu (L), and Cys (C),respectively.

As additional examples, the arginine of the RGD sequence appearing intrigramin, elegantin, albolabrin, crotatroxin, flavoviridin, echistatin,bitistatin, viridin, molossin, lutosin, basilicin, applagin, halysin,horridin, tergeminin, lachesin, cotiarin, and ruberin can be replaced bya K* residue to provide specifically active PAIs with a preferentialaffinity for GP IIb-IIIa. In addition, shortened forms of thesepeptides, containing at least 20, preferably at least 30, and morepreferably at least 40, amino acids, can be prepared from the nativepeptide or in this modified form. In addition, or in the alternative,1-10, preferably 1-4, amino acids irrelevant to the RGD/K GD sequencecan be substituted or modified, preferably with conservative amino acidsubstitutions. By conservative amino acid substitutions is meant, forexample, substitution of an acidic amino acid residue for an acidicamino acid residue, neutral for neutral, basic for basic, etc., as isfurther described hereinbelow.

Still an additional group of examples includes that wherein the glycylresidue of RGD or KGD can be replaced by a sarcosyl residue withretention of activity. Thus, the active PAIs which are isolated and/ormodified in other ways as described above may further be modified bythis substitution.

while fragments and/or modified PAIs from snake venom can be includedamong the Fg/vWF/GP IIb-IIIa binding-specific compounds of the inventionby replacing RGD by K*GD, in additional embodiments of the inventionspecifically active peptides are based on compatible extensions of theK*GD sequence per se. In this regard, a preferred group of peptides orpeptide-related compounds of the invention are cyclic peptides of thegeneral formula: ##STR5## wherein K* is substituted or unsubstitutedlysyl as above defined; AA₁ is a small, neutral (polar or nonpolar)amino acid and n1 is an integer of 0-3;

AA₂ is a neutral, nonpolar large (aromatic or nonaromatic) or a polararomatic amino acid and n2 is an integer of 0-3;

AA₃ is a proline residue or a modified proline residue, and n3 is aninteger of 0-1;

AA₄ is a neutral, small amino acid and n4 is an integer of 0-3;

each of X₁ and X₂ is independently a residue capable of forming a bondbetween X₁ and X₂ to obtain a cyclic compound as shown; and

each of Y₁ and Y₂ is independently a noninterfering substituent or maybe absent;

wherein one or more peptide linkages may optionally be replaced by alinkage selected from the group consisting of --CH₂ NH--, --CH₂ S--, CH₂CH₂ --, --CH═CH-- (cis and trans), --COCH₂ --, --CH(OH)CH₂ -- and --CH₂SO--.

Y₁ and Y₂ can be peptide extensions of 0-25 amino acid residues and maybe in derivatized form. The Y₁ N-terminal extension may, for example, beacetylated or otherwise acylated; the Y₂ C-terminal extension may beamidated with NH₂ or with a primary or secondary amine of the formulaR--NH₂ or R² NH wherein each R is independently a lower alkyl of 1-4Csuch as methyl, n-butyl, or t-butyl. Y₁ can also be (H) or acyl; Y₂ canbe (OH), NH₂ or an amine as above. Where the compound of formula (1) isa simple cyclic peptide, Y₁ and Y₂ are absent.

X₁ and X₂ are typically amino acid residues capable of cyclization suchas, for example and most preferably, cysteine residues capable offorming a disulfide ring. However, other residues capable of formingdisulfide or other linkages may also be used--for example, the Pen(penicillamine) residue described by Pierschbacher et al. (supra) or theMpr (mercapto propionyl) or MVl (mercaptovaleryl) residue at X₁. Othertypes of covalent linkages for cyclization envisioned include peptidelinkages, as for example, an amide formed between the side-chain aminogroup of a lysyl residue with a side-chain carboxyl group of a glutamylresidue and ester linkages, such as would be formed between a side-chainalcohol of a threonine residue with a side-chain carboxyl of an aspartylresidue. Any compatible residue capable of forming peptide bonds withthe remainder of the chain (or modified peptide bonds as describedabove) and capable of covalent bond formation to effect cyclization canbe used. This includes, for example, simple cyclic peptides, wherein apeptide bond is directly formed between the NH₂ at the N-terminus andthe COOH at the C-terminus.

As described above, one or more of the indicated peptide bonds may bereplaced by a substitute linkage such as --CH₂ NH--, --CH₂ S--, CH₂ CH₂--, --CH═CH-- (cis and trans), --COCH₂ --, --CH(OH)CH₂ -- and --CH₂SO--.

In the designation of the amino acid residues AA₁ -AA₄ above,description has been made on the basis of a classification method,wherein amino acid residues can be generally subclassified into fourmajor subclasses. This classification is also shown diagrammaticallyhereinbelow.

Acidic: The residue has a negative charge due to loss of H ion atphysiological pH and the residue is attracted by aqueous solution so asto seek the surface positions in the conformation of a peptide in whichit is contained when the peptide is in aqueous medium at physiologicalpH.

Basic: The residue has a positive charge due to association with H ionat physiological pH and the residue is attracted by aqueous solution soas to seek the surface positions in the conformation of a peptide inwhich it is contained when the peptide is in aqueous medium atphysiological pH.

Neutral/nonpolar: The residues are not charged at physiological pH andthe residue is repelled by aqueous solution so as to seek the innerpositions in the conformation of a peptide in which it is contained whenthe peptide is in aqueous medium. These residues are also designated"hydrophobic" herein.

Neutral/polar: The residues are not charged at physiological pH, but theresidue is attracted by aqueous solution so as to seek the outerpositions in the conformation of a peptide in which it is contained whenthe peptide is in aqueous medium.

It is understood, of course, that in a statistical collection ofindividual residue molecules some molecules will be charged, and somenot, and there will be an attraction for or repulsion from an aqueousmedium to a greater or lesser extent. To fit the definition of"charged", a significant percentage (at least approximately 25%) of theindividual molecules are charged at physiological pH. The degree ofattraction or repulsion required for classification as polar or nonpolaris arbitrary, and, therefore, amino acids specifically contemplated bythe invention have been specifically classified as one or the other.Most amino acids not specifically named can be classified on the basisof known behavior.

Amino acid residues can be further subclassified as cyclic or noncyclic,and aromatic or nonaromatic, self-explanatory classifications withrespect to the side chain substituent groups of the residues, and assmall or large. The residue is considered small if it contains a totalof 4 carbon atoms or less, inclusive of the carboxyl carbon. Smallresidues are, of course, always nonaromatic.

For the naturally occurring protein amino acids, subclassificationaccording to the foregoing scheme is as follows (see also the diagrambelow).

Acidic: Aspartic acid and Glutamic acid;

Basic/noncyclic: Arginine, Lysine;

Basic/cyclic: Histidine;

Neutral/polar/small: Glycine, Serine and Cysteine;

Neutral/polar/large/nonaromatic: Threonine, Asparagine, Glutamine;

Neutral/polar/large/aromatic: Tyrosine;

Neutral/nonpolar/small: Alanine;

Neutral/nonpolar/large/nonaromatic:Valine, Isoleucine, Leucine,Methionine;

Neutral/nonpolar/large/aromatic: Phenylalanine, and Tryptophan.

The gene-encoded amino acid proline, although technically within thegroup neutral/nonpolar/large/cyclic and nonaromatic, is a special casedue to its known effects of the secondary conformation of peptidechains, and is not, therefore, included in this defined group, but isclassified separately. AA₃ is designated a proline residue or a"modified proline residue." Proline, as is understood, is afive-membered nitrogen heterocycle with a carboxyl group in the2-position. Modified proline residues are all nitrogen five orsix-membered heterocycles with carboxyl groups in the position alpha tothe nitrogen; additional heterocyclic atoms may also be included in thering. Thus, modified proline residues include residues of pipecolic acid(2-carboxypiperidine, abbreviated Pip) and thiazolidine (Thz). Thus,proline or modified proline residues are of the formula ##STR6## whereinone or two of the methylene groups may be replaced by NR, S, or O andwhere any ring nitrogen may optionally be substituted with anoninterfering substituent such as alkyl.

Certain commonly encountered amino acids, which are not encoded by thegenetic code, include, for example, beta-alanine (beta-ala), or otheromega-amino acids, such as 3-amino propionic, 4-amino butyric and soforth, alpha-aminoisobutyric acid (Aib), sarcosine (Sar), ornithine(Orn), citrulline (Cit), homoarginine (Har), t-butylalanine (t-BuA),t-butylglycine (t-BuG), N-methylisoleucine (N-MeIle), phenylglycine(Phg), and cyclohexylalanine (Cha), norleucine (Nle), cysteic acid(Cya); pipecolic acid (Pip), thiazolidine (Thz), 2-naphthyl alanine(2-Nal) and methionine sulfoxide (MSO). These also fall convenientlyinto particular categories.

Based on the above definition,

Sar and beta-ala are neutral/nonpolar/small;

t-BuA, t-BuG, N-MeIle, Nle and Cha are neutral/nonpolar/large/nonaromatic;

Har and Orn are basic/noncyclic;

Cya is acidic;

Cit, Acetyl Lys, and MSO are neutral/polar/ large/nonaromatic;

2-Nal and Phg are neutral/nonpolar/large/ aromatic; and

Pip and Thz are modified proline residues.

The foregoing may be shown diagrammatically as follows: ##STR7##

The various omega-amino acids are classified according to size asneutral/nonpolar/small (beta-ala, i.e., 3-aminopropionic,4-aminobutyric) or large (all others).

Other amino acid substitutions for those encoded in the gene can also beincluded in peptide compounds within the scope of the invention and canbe classified within this general scheme.

In the formulas representing selected specific embodiments of thepresent invention, the amino-and carboxy-terminal groups, although oftennot specifically shown, will be understood to be in the form they wouldassume at physiological pH values, unless otherwise specified. Thus, theN-terminal H⁺ ₂ and C-terminal-O⁻ at physiological pH are understood tobe present though not necessarily specified and shown, either inspecific examples or in generic formulas. Of course, the basic and acidaddition salts which are formed at nonphysiological pH values are alsoincluded in the compounds of the invention. Unless otherwise noted, theresidues are in the L-form; in generic formulas, the specified residuescan be either L- or D-. Generally, the peptides of the invention have 0,1, or 2 D-residues included, preferably 0 or 1, most preferably 0. Inthe peptides shown, each encoded residue where appropriate isrepresented by a single letter designation, corresponding to the trivialname of the amino acid, in accordance with the following conventionallist:

    ______________________________________                                        Amino Acid     One-Letter Symbol                                              ______________________________________                                        Alanine        A                                                              Arginine       R                                                              Asparagine     N                                                              Aspartic acid  D                                                              Cysteine       C                                                              Glutamine      Q                                                              Glutamic acid  E                                                              Glycine        G                                                              Histidine      H                                                              Isoleucine     I                                                              Leucine        L                                                              Lysine         K                                                              Methionine     M                                                              Phenylalanine  F                                                              Proline        P                                                              Serine         S                                                              Threonine      T                                                              Tryptophan     W                                                              Tyrosine       Y                                                              Valine         V                                                              Pyroglutamic acid                                                                            Z                                                              ______________________________________                                    

The amino acids not encoded genetically are abbreviated as indicatedabove.

In the specific peptides shown in the present application, the L-form ofany amino acid residue having an optical isomer is intended unlessotherwise expressly indicated by a dagger superscript (.sup.↑). Whilethe residues of the invention peptides are normally in the natural Loptical isomer form, one or two, preferably one, amino acid may bereplaced with the optical isomer D form.

Free functional groups, including those at the carboxy- oramino-terminus, can also be modified by amidation, acylation or othersubstitution, which can, for example, change the solubility of thecompounds without affecting their activity.

In forming amidated peptides of the present invention, the analogcompounds can be synthesized directly, for example using Boc-AA_(x)-pMBHA-Resin or Boc-AA_(x) -BHA-Resin, wherein AA_(x) is the selectedcarboxy-terminal amino acid of the desired peptide as described infurther detail below. Alternatively, the peptides of the presentinvention can be chemically or enzymatically amidated subsequent topeptide synthesis using means well known to the art, or prepared bystandard solution-phase peptide synthesis protocols.

Certain embodiments of the de novo peptides of the invention arepreferred. In the K*(G/Sar)D sequence, G/Sar is preferably G. AA₁ andAA₄ are preferably Gly, Ala or Ser; n1 is preferably 0-2, n4 ispreferably 1-2. Preferred for AA₂ are neutral/nonpolar/aromatic aminoacids, especially tryptophan and phenylalanine, particularly tryptophan,n₂ is preferably 1. X₁ and X₂ are preferably Cys, Mpr, or Pen(penicillamine) residues. Y₁ is preferably H, acetyl, or Gly; Y₂ ispreferably --NH₂ or --A--NH₂. Also preferred generally are C-terminalamidated forms of Y₂.

Thus, preferred embodiments of the PAI analogs of the invention includepeptides of the following formulas. Although all of these may be incyclic form through formation of disulfide linkages, these linkages arenot shown; other cyclic forms are noted by "cyclo."

Preferred Peptides

PAI 1:E-C-A-D-G-L-C-C-D-Q-C-R-F-L-K-K-G-T-V-C-R-V-A-K-G-D-W-N-D-D-T-C-T-G-Q-S-C-D-C-P-R-N-G-L-y-G

PAI 2:E-E-P-C-A-T-G-P-C-C-R-R-C-K-F-K-R-A-G-K-V-C-R-V-A-K-G-D-W-N-N-D-Y-C-T-G-K-S-C-D-C-p-R-N-p-W-N-G

PAI 3: G-C-G-K-G-D-W-P-C-A-NH₂ ;

PAI 4: G-C-K-G-D-W-P-C-A-NH₂

PAI 5: C-G-K-G-D-W-P-C-NH₂

PAI 7: C-K-G-D-W-C-A-NH₂ ;

PAI 9: Mpr-K-G-D-Pen-NH₂

PAI 10: C-K-G-D-W-P-C-NH₂

PAI 12: C-K-G-D-Y-P-C-NH₂

PAI 13: C-K-G-D-F-P-C-NH₂

PAI 14: C-K-G-D-L-P-C-NH₂

PAI 15: C-K-G-D-V-P-C-NH₂

PAI 16: C-K-G-D-Y(OMe)-P-C-NH₂

PAI 17: C-K-G-D-(2-Nal)-P-C-NH₂

PAI 18: C-K-G-D-(Cha)-P-C-NH₂

PAI 19: Mpr-K-G-D-W-P -C-NH₂

PAI 20: Mpr-K-G-D-Y-P-C-NH₂

PAI 21: Mpr-K-G-D-F-P-C-NH₂

PAI 22: Mpr-K-G-D-L-P-C-NH₂

PAI 23: Mpr-K-G-D-V-P-C-NH₂

PAI 24: Mpr-K-G-D-Y(OMe)-P-C-NH₂

PAI 25: Mpr-K-G-D-(2-Nal)-P-C-NH₂

PAI 26: Mpr-K-G-D-(Cha)-P-C-NH₂

PAI 27: cyclo(G-K-G-D-W-P)

PAI 28: cyclo(A-K-G-D-W-P)

PAI 29: cyclo(D-Ala-K-G-D-W-P)

PAI 30: cyclo(F-K-G-D-W-P)

PAI 31: cyclo(beta-Ala-K-G-D-W-P)

PAI 32: cyclo(gamma-Abu-K-G-D-W-P)

PAI 33: cyclo(R-K-G-D-W-P)

PAI 34: C -K-G-D-W-G-C -NH₂

PAI 37: C-K-A-D-W-P-C-NH₂

PAI 39: C-K-G-D-W-(Sar)-C-NH₂

PAI 41: C-K-G-D-I-P-C-NH₂

PAI 42: C-K-G-D-(4-Cl-Phe)-P-NH₂

PAI 43: C-K-(Sar)-D-W-P-C-NH₂

PAI 44: C-K-G-D-(4-NO₂ -Phe)-P-C-NH₂

PAI 47: Acetyl-C-K-G-D-W-P-C-NH₂

PAI 48: Mpr-K-G-D-W(Formyl)-P-C-NH₂

PAI 49: Mvl-K-G-D-W-P-C-NH₂

PAI 51: Mpr-K-G-D-W-P-Pen-NH₂

PAI 52: Mpr-K-G-D-W-P-Pen.sup.↑ -NH₂

PAI 54: Mpr-K-G-D.sup.↑ -W-P-Pen-NH₂

PAI 55: Mpr-K-G-D-W-(Thz)-C-NH₂

PAI 56: Mpr-K-G-D-H(2,4-DNP)-P-C-NH₂

PAI 57: Mpr-K-G-D-(2-Nal)-P-Pen-NH₂

PAI 58: Mvl -K-G-D-W-P-Pen-NH₂

PAI 59: Mpr-K-G-D-W-(Pip)-Pen-NH₂

PAI 60: Mpr-(Har)-G-D-W-P-C-NH₂

PAI 61: Mpr-K-G-D-W-P-C.sup.↑ -NH₂

PAI 62: Mpr-K.sup.↑ -G-D-W-P-Pen-NH₂

PAI 63: Mpr-(Har)-G-D-W-P-Pen-NH₂

PAI 64: Mpr-(Acetimidyl-Lys)-G-D-W-P-C-NH₂

PAI 65: Mpr-(Acetimidyl-Lys)-G-D-W-P-Pen-NH₂

PAI 66: Mpr-(N^(G), N^(G') -ethylene-Har)-G-D-W-P-C-NH₂

PAI 67: Mpr-(N^(G), N^(G') -ethylene-Har)-G-D-W-P-Pen-NH₂

PAI 68: MPr-Har-Sar-D-W-P-C-NH₂

PAI 69: Mpr-(Acetimidyl-lys)-G-D-W-P-Pen-NH₂

PAI 70: Mpr-(Phenylimidyl-lys)-G-D-W-P-C-NH₂

Particularly preferred are peptides of the formulas

PAI 3: G-C-G-K-G-D-W-P-C-A-NH₂ ;

PAI 4: G-C-K-G-D-W-P-C-A-NH₂ ;

PAI 5: C-G-K-G-D-W-P-C-NH₂ ;

PAI 9: Mpr-K-G-D-pen-NH₂ ; and

PAI 10: C-K-G-D-W-P-C-NH₂

PAI 12: C-K-G-D-Y-P -C-NH₂

PAI 13: C-K-G-D-F-D-C-NH₂

PAI 19: Mpr-K-G-D-W-P-C-NH₂

PAI 25: Mpr-K-G-D-(2-Nal)-P-C-NH₂

PAI 34: C-K-G-D-W-G-C-NH₂

PAI 39: C-K-G-D-W-(Sat)-C-NH₂

PAI 42: C-K-G-D-(4-Cl-Phe)-P-NH₂

PAI 43: C-K-(Sar)-D-W-P-C-NH₂

PAI 44: C-K-G-D-(4-NO₂ -Phe)-P-C-NH₂

PAI 47: Acetyl -C-K-G-D-W-P-C-NH₂

PAI 48: Mpr-K-G-D-W(Formyl)-P-C-NH₂

PAI 49: Mvl-K-G-D-W-P-C-NH₂

PAI 51: Mpr-K-G-D-W-P-Pen-NH₂

PAI 52: Mpr-K-G-D-W-P-(D-Pen)-NH₂

PAI 55: Mpr-K-G-D-W-(Thz)-C-NH₂

PAI 56: Mpr-K-G-D-H(2,4-DNP)-P-C-NH₂

PAI 57: Mpr-K-G-D-(2-Nal)-P-Pen-NH₂

PAI 58: Mvl-K-G-D-W-P-Pen-NH₂

PAI 59: Mpr-K-G-D-W-(Pip)-Pen-NH₂

PAI 60: Mpr-(Har)-G-D-W-P-C-NH₂

PAI 61: Mpr-K-G-D-W-P-C.sup.↑ -NH₂

PAI 62: Mpr-K.sup.↑ -G-D-W-p-pen-NH₂

PAI 63: Mpr-(Har)-G-D-W-P-Pen-NH₂

PAI 64: Mpr-(Acetimidyl-Lys)-G-D-W-P-C-NH₂

PAI 65: Mpr-(Acetimidyl-Lys)-G-D-W-P-Pen-NH₂

PAI 66: Mpr (N^(G), N^(G') -ethylene-Har)-G-D-W-P-C-NH₂

PAI 67: Mpr (N^(G),N^(G') -ethylene-Har)-G-D-W-P-Pen-NH₂

PAI 68: Mpr-Har-Sar-D-W-P-C-NH₂

PAI 69: Mpr-(Acetimidyl-Lys)-G-D-W-P-Pen-NH₂

PAI 70: Mpr-(Phenylimidyl-Lys)-G-D-W-P-C-NH₂

Chemical Synthesis of the Invention Peptides

Compounds within the scope of the present invention can be synthesizedchemically by means well known in the art such as, e.g., solid-phasepeptide synthesis. The synthesis is commenced from the carboxy-terminalend of the peptide using an alpha-amino protected amino acid.t-Butyloxycarbonyl (Boc) protective groups can be used for all aminogroups even though other protective groups such asfluorenylmethyloxycarbonyl (Fmoc), are suitable. For example,Boc-Gly-OH, Boc-Ala-OH, Boc-His (Tos)-OH, (i.e., selectedcarboxy-terminal amino acids) can be esterified to chloromethylatedpolystyrene resin supports, p-methyl benzhydrylamine (pMBHA) or PAMresins. The polystyrene resin support is preferably a copolymer ofstyrene with about 0.5 to 2% divinyl benzene as a cross-linking agentwhich causes the polystyrene polymer to be completely insoluble incertain organic solvents. See Stewart, et al., Solid-Phase PeptideSynthesis (1969) W. H. Freeman Co., San Francisco and Merrifield, J AmChem Soc (1963) 85:2149-2154. These and other methods of peptidesynthesis are also exemplified by U.S. Pat. Nos. 3,862,925, 3,842,067,3,972,859, and 4,105,602.

The synthesis may use manual synthesis techniques or automaticallyemploy, for example, an Applied BioSystems 430A or 431A PeptideSynthesizer (Foster City, Calif.) following the instructions provided inthe instruction manual supplied by the manufacturer. Cleavage of thepeptides from the resin can be performed using the "low-high" HFdeprotection protocols as described in Lu, G.-S., et al., Int J Peptide& Protein Res (1987) 29:545-557. Refolding of analogs of the snake venomPAIs can be performed using the procedure outlined in Garsky, V., etal., Proc Natl Acad Sci USA (1989) 86:4022-4026 which describes thesolid-phase synthesis of echistatin.

The cyclic peptides of this invention which do not have disulfide bondscan be conveniently prepared by a combination of solid phase synthesisand formation of the cyclic ring structure in solution using the generalmethods as outlined in U.S. Pat. No. 4,612,366 to Nutt. Thus, linearpeptides prepared on standard Merrifield resin can be cleaved from theresin with hydrazine, followed by cyclization of the corresponding azideto form the cyclic peptides.

It will be readily appreciated by those having ordinary skill in the artof peptide synthesis that the intermediates which are constructed inaccordance with the present disclosure during the course of synthesizingthe present analog compounds are themselves novel and useful compoundsand are thus within the scope of the invention.

Recombinant Production

Alternatively, selected compounds of the present invention can beproduced by expression of recombinant DNA constructs prepared inaccordance with well-known methods. Such production can be desirable toprovide large quantities or alternative embodiments of such compounds.Since the peptide sequences are relatively short, recombinant productionis facilitated.

The DNA encoding the sequenced PAI is preferably prepared usingcommercially available nucleic acid synthesis methods. Methods toconstruct expression systems for production of PAI in recombinant hostsare also generally known in the art.

Expression can be effected in either procaryotic or eucaryotic hosts.Procaryotes most frequently are represented by various strains of E.coli. However, other microbial strains may also be used, such asbacilli, for example Bacillus subtilis, various species of Pseudomonas,or other bacterial strains. In such procaryotic systems, plasmid vectorswhich contain replication sites and control sequences derived from aspecies compatible with the host are used. For example, a workhorsevector for E. coli is pBR322 and its derivatives. Commonly usedprocaryotic control sequences, which contain promoters for transcriptioninitiation, optionally with an operator, along with ribosomebinding-site sequences, include such commonly used promoters as thebeta-lactamase (penicillinase) and lactose (lac) promoter systems, thetryptophan (trp) promoter system, and the lambda-derived P_(L) promoterand N-gene ribosome binding site. However, any available promoter systemcompatible with procaryotes can be used.

Expression systems useful in eucaryotic hosts comprise promoters derivedfrom appropriate eucaryotic genes. A class of promoters useful in yeast,for example, includes promoters for synthesis of glycolytic enzymes,e.g., those for 3-phosphoglycerate kinase. Other yeast promoters includethose from the enolase gene or the Leu2 gene obtained from YEp13.

Suitable mammalian promoters include the early and late promoters fromSV40 or other viral promoters such as those derived from polyoma,adenovirus II, bovine papilloma virus or arian sarcoma viruses. Suitablevital and mammalian enhancers are cited above. In the event plant cellsare used as an expression system, the hopaline synthesis promoter, forexample, is appropriate.

The expression systems are constructed using well-known restriction andligation techniques and transformed into appropriate hosts.

Transformation is done using standard techniques appropriate to suchcells. The cells containing the expression systems are cultured underconditions appropriate for production of the PAI, and the PAI is thenrecovered and purified.

Antibodies

The availability of the purified PAI of the invention also permits theproduction of antibodies specifically immunoreactive with these forms ofthe active peptide.

The compositions containing purified PAI isolated from snake venom orotherwise synthesized can be used to stimulate the production ofantibodies which immunoreact with the PAI peptide. Standard immunizationprotocols involving administering PAI to various vertebrates, such asrabbits, rats, mice, sheep, and chickens result in antisera which areimmunoreactive with the purified peptide. PAI may be advantageouslyconjugated to a suitable antigenically neutral carrier, such as anappropriate serum albumin or keyhole limpet hemocyanin, in order toenhance immunogenicity. In addition, the free peptide can be injectedwith methylated BSA as an alternative to conjugation. Furthermore, theantibody-secreting cells of the immunized mammal can be immortalized togenerate monoclonal antibody panels which can then be screened forreactivity with PAI.

The resulting polyclonal or monoclonal antibody preparations are usefulin assays for levels of the corresponding PAI in biological samplesusing standard immunoassay procedures.

The Invention Assay

The identification of snake venom starting material which containsactive PAI, and which PAI has known specificity, is made possible by theassay of the invention. The assay rests on the observation thatcompounds which block the binding of fibrinogen to the GP IIb-IIIacomplex in vitro also are capable of inhibiting thrombin or ADP-inducedaggregation of human platelets and the formation of platelet-thrombi invivo. This observation provides the basis for obtaining potent PAI byevaluating the ability of test materials to disrupt fibrinogen-GPIIb-IIIa interactions.

In the assay, GP IIb-IIIa, prepared in purified form, for example asdescribed by Fitzgerald, L. A., et al., Anal Biochem (1985) 151:169-177,incorporated herein by reference, is coated onto a solid support such asbeads, test tubes, or microtiter plates. The coated support is thencontacted with fibrinogen and with the test material and incubated for asufficient time to permit maximal binding of fibrinogen to theimmobilized GP IIb-IIIa. Fibrinogen is typically provided at aconcentration of about 5-50 nM and the test material can, if desired, beadded at a series of dilutions. Typical incubations are 2-4 hr at 35°C., the time and temperature being interdependent.

After incubation, the solution containing the fibrinogen and testmaterial is removed and the level of binding of fibrinogen measured byquantitating bound fibrinogen to GP IIb-IIIa. Any suitable means ofdetection may be used, but it is convenient to employ labeledfibrinogen, for example using radioactive, fluorescent or biotinylatedlabels. Such methods are well known and need not be elaborated here.

Assessment of the results is aided by employing a control sample,usually identical to the test sample except that the test substance isabsent. In this case, percent inhibition may be calculated using th&amount of Fg bound in the control as representing the basis, so that##EQU1## Other measures of inhibition effectiveness, such as IC₅₀, mayalso be used.

The assay systems of the invention further include characterization ofthe PAI specificity by binding inhibition assays identical to that abovebut substituting other adhesive proteins for Fg and other receptors forGP IIb-IIIa. In particular, inhibition of the binding of vitronectin tothe vitronectin receptor; fibronectin to the fibronectin receptor;fibronectin to GP IIb-IIIa and fibrinogen and/or vWF to GP IIb-IIIa maybe assessed. The adhesive protein and receptors for these assays areavailable in the art.

Other Assays

In addition to the plate assays of the invention, other assays forplatelet aggregation inhibition activity and related activities are alsoavailable, as set forth above. In summary, a list of commonly employedassays is as follows:

1. The plate assays utilizing specific receptors described in theprevious paragraphs;

2. Standard assays directly applied to platelet aggregation, such asthose described by Gann, Z.-R., et al., J Biol Chem (1988)263:19827-19832; Huang, T. F., et al., J Biol Chem (1987)262:16157-16163; Biochemistry (1989) 28:661-666, cited above andincorporated herein by reference;

3. An in vivo thrombosis model in dogs as described hereinbelow inExample 1, and by Folts, J. D., et al., Circulation (1976) 54:365; and

4. Effect on cell adhesion using S35 methionine-labeled cells asdescribed hereinbelow in Example 19.

Administration and Utility

The PAIs of the invention are useful therapeutically to prevent thrombusformation. Indications appropriate to such treatment include, withoutlimitation, atherosclerosis and arteriosclerosis, acute myocardialinfarction, chronic unstable angina, transient ischemic attacks andstrokes, peripheral vascular disease, arterial thrombosis, preeclampsia,embolism, restenosis and/or thrombosis following angioplasty, carotidendarterectomy, anastomosis of vascular grafts, and chroniccardiovascular devices (e.g., in-dwelling catheters or shunts"extracorporeal circulating devices"). These syndromes represent avariety of stenotic and occlusive vascular disorders thought to beinitiated by platelet activation on vessel walls.

The PAIs may be used for prevention or abortion of arterial thrombusformation, in unstable angina and arterial emboli or thrombosis, as wellas treatment or prevention of myocardial infarction (MI) and muralthrombus formation post MI. For brain-related disorders, treatment orprevention of transient ischemic attack and treatment of thromboticstroke or stroke-in-evolution are included.

The PAIs may also be used for prevention of platelet aggregation,embolization, or consumption in extracorporeal circulations, includingimproving renal dialysis, cardiopulmonary bypasses, hemoperfusions, andplasmapheresis.

PAIs prevent platelet aggregation, embolization, or consumptionassociated with intravascular devices, and administration results inimproved utility of intraaortic balloon pumps, ventricular assistdevices, and arterial catheters.

The PAIs will also be useful in treatment or prevention of venousthrombosis as in deep venous thrombosis, IVC, renal vein or portal veinthrombosis, and pulmonary venous thrombosis.

Various disorders involving platelet consumption, such as thromboticthrombocytopenic purpura are also treatable.

In addition, the PAIs of the present invention may be used in numerousnontherapeutic applications where inhibiting platelet aggregation isdesired. For example, improved platelet and whole blood storage can beobtained by adding sufficient quantities of the peptides, the amount ofwhich will vary depending upon the length of proposed storage time, theconditions of storage, the ultimate use of the stored material, etc.

The PAI dosage can range broadly depending upon the desired affects andthe therapeutic setting. Typically, dosages will be between about 0.01and 10 mg/kg, preferably between about 0.01 to 0.1 mg/kg, body weight.Administration is preferably parenteral, such as intravenous on a dailybasis for up to a week or as much as one or two months or more, all ofwhich will vary with the peptide's size. If the peptides aresufficiently small (e.g., less than about 8-10 amino acid residues)other routes of administration can be utilized, such as intranasally,sublingually, or the like.

Injectables can be prepared in conventional forms, either as liquidsolutions or suspensions, solid forms suitable for solution orsuspension in liquid prior to injection, or as emulsions. Suitableexcipients are, for example, water, saline, dextrose, mannitol, lactose,lecithin, albumin, sodium glutamate, cysteine hydrochloride or the like.In addition, if desired, the injectable pharmaceutical compositions maycontain minor amounts of nontoxic auxiliary substances, such as wettingagents, pH buffering agents, and the like. If desired, absorptionenhancing preparations (e.g., liposomes) may be utilized.

EXAMPLE 1 Assay for Snake Venom Platelet Adhesion Inhibitors

A. Description of Assays--Plate Assays

Purified platelet GP IIb-IIIa receptor was prepared as described byFitzgerald, L. A., et al., Anal Biochem (1985) 151:169-177. Vitronectinreceptor was prepared as described by Smith, J. W., J Biol Chem (1988)263:18726-18731. After purification, the receptors were stored in 0.1%Triton X-100 at 0.1-1.0 mg/ml.

The receptors were coated to the wells of 96-well flat-bottom ELISAplates (Linbro EIA-Plus microtiter plate, Flow Laboratories) afterdiluting 1:200 with a solution of 20 mM Tris-HCl, 150 mM NaCl, 1 mMCaCl₂, pH 7.4, to reduce the Triton X-100 concentration to below itscritical micellar concentration and adding an aliquot of 100 ul to eachwell. The wells were all allowed to incubate overnight at 4° C., andthen aspirated to dryness. Additional sites were blocked by the additionof bovine serum albumin (BSA) at 35 mg/ml in the above buffer for 2 hrat 30° C. to prevent nonspecific binding. The wells were then washedonce with binding buffer (50 nM Tris-HCl, 100 mM NaCl, 2 mM CaCl₂, 1mg/ml BSA).

The corresponding ligands (fibrinogen, von Willebrand Factor, orvitronectin) were labeled with ¹²⁵ I or conjugated to biotin usingcommercially available reagents and standard protocols. The labeledligands were added to the receptor-coated wells at final concentrationof 10 nM (100 ul/well) and incubated for 3 h at 30° C. in the presenceor absence of the test samples. After incubation, the wells areaspirated to dryness and bound ligand is quantitated.

For ¹²⁵ I-labeled ligands, the protein is solubilized with 250 ul SDS.For biotinylated ligands, the bound protein is detected by the additionof antibiotin antibody conjugated to alkaline phosphatase followed byaddition of substrate (p-nitrophenyl phosphate), and determination ofthe optical density of each well at 405 nm. Decreased color developmentor decreased ¹²⁵ I content is observed in wells incubated with testsamples which inhibit binding of ligand to receptor.

B. Determination of Adhesion Inhibition in Crude Venom

Sixty-eight crude, lyophilized snake venoms obtained from either SigmaChemical Company (St. Louis, Mo.) or Miami Serpentarium Labs (Salt LakeCity, Utah) were dissolved at 1 mg/ml in buffer (50 mM Tris, 100 mMNaCl, 0.02% azide, 2 mM CaCl₂). One ml aliquots of the solutions weresubjected to ultrafiltration through Centrocon-10 (YM membrane)microconcentrators (Amicon, Danvers, Mass.). The filtrates were used astest samples in the receptor/ligand assay of paragraph A using the GPIIb-IIIa/fibrinogen system, and detecting binding using biotinylatedfibrinogen. The results are shown in Table 1.

It is seen that the activity is present in some, but not all, species ofViperinae, but absent in all species tested of Elapidae.

FIG. 1 shows the results at various dilutions of the filtrate for fourspecies. Even at the greatest dilution, 25 ul/0.5 ml, the three activevenoms showed maximal inhibition.

C. Determination of the Activity of Peptides in an in vivo Model ofThrombosis

Purified peptides were tested for their ability to prevent thrombiformation in dog coronary arteries in the model described by Folts(Folts J. D., et al., Circulation (1976) 54:365. In this model, flowreductions in a constricted coronary artery have been shown to be due tothe formation of platelet aggregates, and agents which block the bindingof fibrinogen to GP IIb-IIIa have been shown to prevent these flowreductions (Coller, B. S., et al., Blood (1986) 68:783. The peptideswere dissolved in normal saline and administered into a peripheral veinas a single bolus.

                  TABLE 1                                                         ______________________________________                                        CENTRICON 10 PURIFIED VENOMS SCREENED IN                                      IIb-IIIa PLATE ASSAY                                                                                 Activity                                               ______________________________________                                        Elapids                                                                       Austrelaps superba (Austraiian Copperhead)                                                             -                                                    Acanthopis antarcticus (Death Adder)                                                                   -                                                    Dendroaspis jamesonii (Jameson's Mamba)                                                                -                                                    Notechis scutatus (Mainland Tiger)                                                                     -                                                    Pseudechis colleti guttatus (Blue-bellied)                                                             -                                                    Pseudechis textillis textillis (Common Brown)                                                          -                                                    Oxyuranus scutellatus (Papuan Taipan)                                                                  -                                                    Viperinae (True Vipers)                                                       atheris squamigera (Green Bush Viper)                                                                  -                                                    Bitis nasicornus (River Jack)                                                                          -                                                    Causus rhombeatus (Rhombic Night Adder)                                                                -                                                    Cerastes cerastes (Desert Horned Viper)                                                                -                                                    Cerastes vipera (Sahara Horned Viper)                                                                  -                                                    Echis carinatus (Saw-scaled Viper)                                                                     +                                                    Echis colorata (Carpet Viper)                                                                          +                                                    Eristicophis macmahonii (Macmahons Viper)                                                              ++                                                   Pseudocerastes fieldi (Persian Horned Viper)                                                           -                                                    Vipera xanthina xanthina (Ottomans Viper)                                                              -                                                    Vipera ammodytes (Long-nosed Viper)                                                                    -                                                    Vipera r. russelli (Russells Viper)                                                                    -                                                    Vipera r. siamensis      -                                                    Vipera palaestinae (Palestine Viper)                                                                   -                                                    Crotalinae (Pit Vipers)                                                       Agkistrodon rhodostoma (Malayan Pit Viper)                                                             +                                                    Agkistrodon halys blomhoffi (Mamushi)                                                                  +                                                    Agkistrodon hypnale (Hump-nosed Viper)                                                                 +                                                    Agkistrodon acutus (Sharp-nosed Viper)                                                                 ++                                                   Agkistrodon bilineatus (Mexican Moccasin)                                                              -                                                    Agkistrodon contortrix contortrix                                                                      -                                                    Agkistrodon c. laticinctus                                                                             -                                                    Agkistrodon c. pictigaster                                                                             -                                                    Agkistrodon contortrix mokasen (Northern                                                               -                                                    Copperhead)                                                                   Agkistrodon piscivorous piscivorous                                                                    -                                                    (Eastern Cottonmouth)                                                         Agkistrodon piscivorus leucostoma                                                                      +                                                    (Western Cottonmouth)                                                         Agkistrodon piscivorous conanti                                                                        +                                                    Bothrops asper           +                                                    Bothrops nummifer (Jumping Viper)                                                                      -                                                    Bothrops cotiara (Cotiara)                                                                             +                                                    Bothrops jararacussu (Jararacussu)                                                                     +                                                    Bothrops jararaca (Jararaca)                                                                           +                                                    Bothrops lansbergi       +                                                    Bothrops alternata (Urutu)                                                                             -                                                    Bothrops medusa          +                                                    Bothrops neuwiedi        +                                                    .sbsp.Bothrops nasuta    +                                                    Bothrops pradoi          +                                                    Bothrops schlegli (Schlegels Viper)                                                                    -                                                    Trimeresurus gramineus (Formosan Green Habu)                                                           -                                                    Trimeresurus flavoviridis (Okinawa Habu)                                                               -                                                    Trimeresurus wagleri     -                                                    Lachesis mutas (Bushmaster)                                                                            -                                                    Crotalus durrisus terrificus                                                                           -                                                    (Tropical Rattlesnake                                                         Crotalus durissus totonatacus                                                                          -                                                    Crotalus durissus durissus                                                                             +                                                    Crotalus scutalatus (Mojave rattlesnake)                                                               -                                                    Cortalus horridus horridus (Timber Rattlesnake)                                                        +                                                    Crotalus horridus atricaudatus (Canebrake RS)                                                          -                                                    Crotalus atrox (Western Diamondback)                                                                   -                                                    Crotalus adamanteus (Eastern Diamondback)                                                              -                                                    Crotalus basilicus (Mexican West-coast RS)                                                             +                                                    Crotalus molossus molossus (Black-tailed RS)                                                           +                                                    Crotalus ruber ruber (Red diamondback RS)                                                              +                                                    Crotalus cerastes cerastes (Mojave sidewinder)                                                         +                                                    Crotalus viridis viridis (Prairie Rattlesnake)                                                         +                                                    Crotalus v. helleri (Southern pacific RS)                                                              +                                                    Crotalus v. oreganus (Northern pacific RS)                                                             +                                                    Crotalus v. cereberus (Arizona black RS)                                                               +                                                    Crotalus v. lutosus (Great Basin RS)                                                                   +                                                    Crotalus v. concolor (Midget-faded RS)                                                                 -                                                    Sistrurus catenatus tergeminus                                                                         +                                                    (Western massasauga)                                                          Sistrurus milarius barbouri                                                                            +                                                    (Southeastern Pigmy Rattlesnake)                                              ______________________________________                                    

D. Effects of Purified Snake Venom Peptides on Cell Attachment toAdhesive Proteins

M21 melanoma cells, which express high levels of the vitronectinreceptor, were metabolically labelled with ³⁵ S-methionine, and thenadded to 24-well tissue culture plates coated with vitronectin. Anincubation period of 1 hr at 37° C. was allowed for cell attachment, andthis was followed by a wash to remove non-adherent cells. After washing,the adherent cells were solubilized, and the supernatants placed in aliquid scintillation counter. The fraction of cells remaining adherentwas calculated by dividing the cpm in the solubilized supernatants bythe cpm in the total number of cells added to each well. The effects ofpurifed snake venom peptides and synthetic cyclic peptides on celladhesion was determined by including them with the M21 cells during theincubation period.

E. Specificity of Adhesion Inhibition

Ultrafiltrates from three species of snake venom, Sistrurus m. barbouri,Crotalus ruber ruber, and Crotalus basilicus, were tested in both thefibrinogen/GP IIb-IIIa and vitronectin/vitronectin receptor assays ofparagraph A. The results were evaluated at various dilutions. As shownin FIG. 2A, the venom from Sistrurus m. barbouri preferentially inhibitsthe binding of fibrinogen to GP IIb-IIIa; as shown in FIG. 2B the venomof Crotalus ruber ruber inhibits binding in both systems approximatelyequally; and as shown in FIG. 2C the venom from Crotalus basilicuspreferentially inhibits vitronectin/vitronectin receptor binding.

In the purifications described in Examples 2-6 and 8-12, PAI activitywas assayed using a direct inhibition of platelet aggregation. Plateletrich plasma (PRP) was obtained from a healthy human volunteer.Aggregation was induced by the addition of 4 uM ADP to 0.5 ml PRP in anaggregometer (Chrono-log Corp.).

A table showing results of amino acid composition analysis of purifiedPAIs of Examples 2-6 will be found after Example 6; that showing theresults for Examples 8-11 is shown after Example 8.

This analysis was obtained by hydrolysis of peptides using 6 N HCl andanalyzing the hydrolysate using a Beckman 121HC analyzer equipped with aModel 126 data system. Cysteic acid was determined according to themethod of Moore, J Biol Chem (1969) 230:235-237. Tryptophan was notdetermined.

EXAMPLE 2 Purification of Platelet Aggregation Inhibitor (PAI) FromEristocophis macmahoni Venom

A solution of 45 mg of Eristocophis macmahoni venom (Miami SerpentariumLabs, Lot #EM23SZ) in 1.0 ml of 0.5% trifluoroacetic acid (TFA) wascooled on ice for 20 min, spun at 14,000 rpm for 3 min to removeinsoluble material and loaded onto a 3.9 mm×30 cm, C-18 Delta Pakreverse-phase HPLC column (Waters, Milford, Mass.) equilibrated with 5%acetonitrile containing 1% TFA. A gradient running from 5% to 15%acetonitrile over 5 min (2%/min) followed by a gradient from 15% to 30%acetonitrile over 35 min and then to 50% acetonitrile over 20 min, wasrun using a Waters 600E liquid chromatograph. A flow rate of 1.5 ml/minwas maintained throughout the gradient and column effluent was collectedin 2 min fractions into polypropylene tubes.

The column effluent was monitored at 220 nm/2.5 absorbance units fullscale (AUFS).

Fractions were concentrated to one-half their original volume using aSpeed-Vac concentrator (Savant) followed by lyophilization. Samples werethen reconstituted in 1 ml distilled water and aliquots (10-50 ul)assayed for their ability to inhibit human platelet aggregation inplatelet-rich plasma induced by 20 uM ADP using a whole bloodaggregometer (Chrono-Log Corp., Havertown, Pa.).

As shown in FIG. 3, activity was found in fractions that eluted at21-25% acetonitrile concentration. These fractions were then lyophilizedand rerun on the C-18 HPLC column using shallower acetonitrile gradientas follows: Initial conditions consisted of 8% acetonitrile followed bya gradient to 25% acetonitrile over 68 min (0.25%/min), then to 60%acetonitrile in 10 min. One-minute fractions were collected, dried andreassayed for inhibitory activity in platelet aggregation of humanplatelets as above.

As shown in FIG. 4, the activity eluted at 24% acetonitrile. The activefractions were then subjected to analytical HPLC with detection at 220nm and eluted as a single symmetric bioactive component as shown in FIG.5. Amino acid analysis of the HPLC-purified material showed that thepeptide contains 49 residues including 7-8 cysteines, as set forth inTable 2.

Attempts at automated Edman degradation of the carboxyamidomethylatedpeptide did not yield any detectable sequence. Therefore, digestion ofthis material was performed with Lys-C and Asp-N endoproteinasesyielding fragments which were sequenced and are shown in FIG. 6. Thisanalysis revealed a sequence of 48 residues. However, since twotryptophan residues are apparent from this sequence analysis which werenot determined in the amino acid composition, the intact peptidecontains 51 amino acid residues. Thus, two Glx and one Arg residuesmissing from the determined sequence were presumably present at theblocked amino terminus of the peptide. Since it was quite likely thatone of the Glx residues was a pyroglutamyl residue at the amino terminusleading to the blocked nature of the intact peptide, we removed thisgroup from the intact, carboxyamidomethylated peptide with the enzymepyroglutamyl aminopeptidase (L-pyroglutamyl peptide hydrolase, EC3.4.11.8, Boehringer Mannheim Biochemicals, Indianapolis, Ind.).Protocols described by Podell and Abraham, Biochem Biophys Res Commun(1978) 81:176-185 were used. Digestion of 100 ug of peptide with thepeptidase at a substrate-to-enzyme ratio of 100:1, followed byreversed-phase HPLC purification of the mixture on a Waters analyticalC-18 column gave material suitable for automated Edman degradation. Theresults of this analysis and the assignment of the entire sequence ofthis peptide which was named "eristicophin," is shown in FIG. 6-1.

The complete amino acid sequence of this PAI, is shown in FIG. 6. Thispeptide has RGD in the binding region and shows considerable homology toechistatin.

EXAMPLE 3 Purification of PAI from Sistrurus catenatus tergeminus Venom

Three hundred sixty mg of Sistrurus c. tergeminus venom (MiamiSerpentarium Labs, Lot #ST6SZ) was dissolved in 7.0 ml of 0.5M aceticacid and applied to a column of Sephadex G-50 fine (Pharmacia, 2.5×100cm) equilibrated and eluted with 0.5M acetic acid. The column was run ata flow rate of approximately 25 ml/hr and 5-ml fractions collected.Twenty-five ul of each fraction was pooled in groups of 10 fractions(i.e., fractions 1-10, 11-20, etc.) and lyophilized for analysis. Thedried pooled fractions were redissolved in water and aliquots assayedfor inhibitory activity in ADP-stimulated aggregation of humanplatelets. Active fractions (31-40) were pooled and lyophilized.

This material was dissolved in 2 ml of 0.5% TFA and loaded onto a 19mm×30 cm C-18 Delta Pak reverse-phase HPLC column (Waters) equilibratedwith 8% acetonitrile containing 0.1% TFA. A gradient from 8% to 30%acetonitrile concentration over 30 min and then to 60% acetonitrile overtwenty min was run at a flow rate of 18 ml/min. The column effluent wascollected into polypropylene tubes in 0.2 min fractions and monitored at220 nm/2.2 AUFS. Fractions were concentrated on a Speed-Vac concentrator(Savant), lyophilized and assayed for antiaggregation activity withhuman platelets as previously described.

FIG. 7 shows that the PAI-containing fraction elutes at 24-25%acetonitrile. Analysis of these active fractions using HPLC withdetection at 220 nm showed a symmetric bioactive component, as shown inFIG. 8. The amino acid analysis of this material showed a peptide of71-72 residues, including 12 cysteines, as shown in Table 2.

A portion of the purified peptide was reduced and alkylated withiodoacetamide and purified on a C-18 reverse-phase HPLC column.N-terminal sequence analysis of this material revealed the followingamino acid sequence for 23 cycles of Edman degradation:Glu-Ala-Gly-Glu-Glu-Cys-Asp-Cys-Gly-Ser-Pro-Ala-Asn-Pro-Cys-Cys-Asp-Ala-Ala-Thr-Cys-Lys-Leu.

The complete amino acid sequence for this PAI, which was named"tergeminin" is shown in FIG. 6-1.

The purified peptide was tested in the receptor-based assays describedin Example 1, paragraph A. Concentrations of pure peptide at less than100 nM inhibited the binding of Fg and vWF to GP IIb-IIIa and of Vn andvWF to the vitronectin receptor, as shown in FIG. 9.

EXAMPLE 4 Purification of Platelet Aggregation Inhibitor from Sistrurusmilarus barbouri Venom

Two hundred mg of Sistrurus m. barbouri venom (Miami Serpentarium Labs,Lot #SM13SZ) was dissolved in 7.0 ml of 0.5M acetic acid and applied toa column of Sephadex G-50 fine (Pharmacia, 2.5×100 cm) equilibrated andeluted with 0.5M acetic acid. The column was run at a flow rate of 26ml/hr and 5 ml fractions were collected and analyzed for antiplateletaggregation activity as previously described. Active fractions (41-50)were pooled and lyophilized. This material was redissolved in 2.0 ml0.5% TFA and loaded onto the preparative C-18 HPLC column as in Example3 and eluted employing the same gradient conditions. Two-tenths-minfractions from the column were collected into polypropylene tubes,concentrated, lyophilized and analyzed for platelet aggregationinhibitory activity.

FIG. 10 shows the activity profile from this HPLC column. The activefractions were subjected to analytical HPLC, which showed severalfractions (45-47) which were more than 90% homogeneous. The peptide offraction 46 (150 ug) was purified to homogeneity on an analytical C-18column with manual collection of the symmetric peak, as shown in FIG.11. Amino acid analysis of this material showed a peptide of 71-72 aminoacids, including 12 cysteine residues, as set forth in Table 2.

The purified peptide (150 ug) was dissolved in 300 ul reaction buffer(6M guanidine HCl, 0.25M Tris-HCl, 20 mM EDTA, 20 mM dithiothreitol(DTT), pH 7.5) for 1.5 hours at room temperature to reduce the peptide.This was followed by reaction of 3 ul of 4-vinylpyridine (Aldrich) atroom temperature for an additional hour. The reaction was stopped byaddition of 200 ul 1% TFA and loaded onto an analytical C-18 HPLC columnand eluted with an acetonitrile gradient in water containing 0.1% TFA,starting at 8% acetonitrile and running to 25% acetonitrile in 20minutes, then to 60% acetonitrile in 10 minutes.

A portion of this pyridylethylated material was submitted to N-terminalsequence analysis, as described above, and exhaustive proteolyticcleavage of the reduced and alkylated peptide was performed usingendoproteinase Lys-C and endoproteinase Asp-N with peptide fragmentsisolated on either C-3 or C-18 reverse-phase HPLC columns usingacetonitrile/water/TFA gradient elution. The amino acid sequence of theN-terminus of the intact peptide and isolated proteolytic fragments weredetermined as described by Yarden, Y., et al., Nature (1986) 323:226,using automated Edman degradation on a gas-phase sequencer.

The complete amino acid sequence of this isolated peptide, designated"barbourin" is shown in FIG. 6-1, along with the sequences for theproteolytic fragments. A comparison of this sequence with those of othersnake venom adhesion inhibitors is shown in FIG. 12.

EXAMPLE 5 Purification of PAI from Lachesis mutas venom

99 mg of Lachesis mutas venom (Miami Serpentarium Labs, Lot #LM15FZ) wasdissolved in 2.0 ml of 0.5% trifluoroacetic acid was cooled on ice for20 min, spun at 14,000 rpm for 3 min to remove insoluble material andloaded onto a 3.9 mm×30 cm, C-18 Delta Pak reversed-phase HPLC column(Waters) equilibrated with 5% acetonitrile containing 0.1%trifluoroacetic acid. A gradient form 5% to 15% acetonitrile over 5 minand then to 30% over 35 min (2%/min) and continued to 60% acetonitrileover 20 min was run. The flow rate was maintained at 1.5 ml/min and thecolumn effluent monitored at 220 nm/3.0 AUFS. Two minute fractions werecollected, concentrated by Speed-vac and lyophilized. Fractions wereassayed for platelet aggregation inhibitory activity.

FIG. 13 shows the active fractions which elute at 18% acetonitrile.These fractions were rerun on the C-18 column using a shallower gradientconsisting of a 40 min gradient from 5-28% acetonitrile. One-minfractions were collected, concentrated, lyophilized and assayed forplatelet aggregation inhibition activity, with the results shown in FIG.14. These active fractions were run on an analytical C-18 column, andthe eluted center peak fraction collected by hand. The eluted material,which is in a single symmetric peak, as shown in FIG. 15, was subjectedto amino acid analysis and showed a peptide of 72-73 amino acidscontaining 12 cysteines, as shown in Table 2.

The complete amino acid sequence of this PAI, called "lachesin" is shownin FIG. 6-2.

EXAMPLE 6 Purification of PAI from Crutalus viridis viridis Venom

47 mg of Crotalus viridis viridis venom (Sigma Chemical Co., Lot#24F-0534) was dissolved in 1 ml of 0.5% trifluoroacetic acid, cooled onice for 20 min, spun at 14,000 rpm for 3 min to remove insolublematerial and loaded onto a 3.9 mm×30 cm C-18 Delta Pak reverse-phaseHPLC column (Waters) equilibrated with 5% acetonitrile containing 0.1%trifluoroacetic acid. A gradient from 5% to 15% acetonitrile over 5 min(2%/min) followed by a gradient from 15% to 30% acetonitrile in 35 minand then to 60% acetonitrile in 60 min was run. A flow rate of 1.5ml/min was maintained throughout the gradient and the column effluentwas collected into polypropylene tubes in 2 min fractions. The columneffluent was monitored at 220 nm/3.0 AUFS. Fractions were concentrated,lyophilized and assayed for platelet aggregation inhibitory activity.

The active fractions, shown in FIG. 16 as 18-19% acetonitrile, were runon the C-18 HPLC column using a gradient of 8%-20% acetonitrile over 48min (0.25%/min). The fractions were concentrated and lyophilized andtested for activity; the active fractions were run on a C-18 columnusing 8-16% acetonitrile over 10 min, 16-20% acetonitrile over 15 min,and then to 60% over 10 min. The effluent was monitored at 220 nm withindividual peaks collected by hand into polypropylene tubes. Reanalysisof the active peak on analytical HPLC gave the results shown in FIG. 17.The amino acid analysis conducted on this peak showed a 72-73-residuepeptide containing 12 cysteines, as set forth in Table 2. The completeamino acid sequence of this PAI called "viridin" is shown in FIG. 6-3and is compared to other PAI in FIG. 12-1.

                                      TABLE 2                                     __________________________________________________________________________    Amino acid Compositions of Purified Peptides                                  Amino                                                                             Sistrurus                                                                            Sistrurus                                                                             Lachesis                                                                            Crotalus                                                                           Eristicophis                                    acid                                                                              m. barbouri                                                                          c. tergeminus                                                                         mutas v. viridis                                                                         macmahoni                                       __________________________________________________________________________    Lys 4      3       4     3-4  4                                               His 0      0       0-1   1    0                                               Arg 4      5       7     5    7                                               Asx 11     11      10    11   7                                               Thr 4      4       2     4    2                                               Ser 2      2       1     2    1                                               Glx 6-7    5-6     7     6    4                                               Pro 4      4       5     6    5                                               Gly 9      9       9     10   5                                               Ala 7      8       9     7    3                                               Cys 12     12      12    12   7                                               Val 2      2       0     1    2                                               Met 1      1       0     0    0                                               Ile 0      0       2     1    0                                               Leu 3      3       2     3    0                                               Tyr 1      1       1     1    1                                               Phe 1      1       1     1    1                                                   71-72  71-72   72-73 74-75                                                                              49                                              __________________________________________________________________________

EXAMPLE 7 Comparison of Purified PAI to Echistatin

The peptides purified as described in Examples 2 and 4, eristicophin andbarbourin, were compared to the 49-residue peptide echistatin ininhibiting fibrinogen binding to GP IIb-IIIa, as described in Example 1,paragraph A. FIG. 18 shows that these purified PAIs are 2-3 times morepotent in this assay than the standard echistatin.

Peptides purified to homogeneity from Echis carinatus, Sistrurus m.barbouri, and Eristicophis macmahoni venoms were compared to echistatinin the ADP-stimulated platelet aggregation assay. Increasingconcentrations of purified snake venom peptides were added (withoutpreincubation) at the indicated concentrations (FIGS. 19A, 19B, and19C). Snake venom peptides from Eristicophis macmahoni and Sistrurus m.barbouri were at least twofold more potent than echistatin, in agreementwith their order of potency observed for inhibiting fibrinogen bindingto GP IIb-IIIa as presented above.

EXAMPLE 8 Purification of PAI from Crotalus cerastes cerastes Venom

One gram of Crotalus c. cerastes venom (Miami Serpentarium Labs, Lot#CE4SZ) was dissolved in 7.0 ml of 0.5M acetic acid and applied to acolumn of Sephadex G-50 fine (Pharmacia, 2.5×100 cm) equilibrated andeluted with 0.5M acetic acid. The column was run at a flow rate of 25ml/hr with 5 ml fractions collected into polypropylene tubes. Aliquotsof these fractions were assayed for aggregation activity inhibitoryactivity as previously described. Active fractions (71-80) were pooledand lyophilized. The dried material was resuspended in 2.0 ml of 0.5%TFA, insoluble material removed by centrifugation and loaded onto thepreparative C-18 Waters HPLC column as described in Example 3 and elutedemploying the gradient elution conditions described in Example 3.Fractions from the column were collected into polypropylene tubes,concentrated and analyzed for platelet aggregation inhibitory activity.FIG. 20 shows the activity profile from this HPLC fractionation.

Active fractions with platelet aggregation inhibitory activity werepooled and lyophilized and rerun on the preparative C-18 HPLC columneluted with the same gradient. Fractions were collected by hand intopolypropylene tubes and again assayed for platelet aggregationinhibitory activity as before. Active fractions were analyzed on ananalytical C-18 column using the conditions described in Example 4 andhomogeneous fractions were pooled and lyophilized. Analytical HPLCanalysis of this material is shown in FIG. 21.

Purified peptide was subjected to amino acid analysis revealing that itwas a peptide of 73-74 amino acids containing 12 cysteine residues, asset forth in Table 3.

The purified peptide (450 ug) was dissolved in 750 ul reaction buffer(6M guanidine-HCl, 0.25M Tris-HCl, 20 mM EDTA, 20 mM dithiothreitol(DTT), pH 7.50) for 1.5 hr at room temperature to fully reduce thepeptide followed by reaction at room temperature for 1 hr with excessiodoacetamide (Fluka, 16 mg). The reaction was stopped by addition of500 ul of 1% TFA and loaded onto an analytical C-18 HPLC column andeluted with a gradient of acetonitrile from 8% to 25% in 20 minutes,then to 60% acetonitrile in 10 minutes. The UV absorbing peak wascollected by hand into 1.5 ml eppendorf tubes and dried.

A portion of this carboxyamidomethylated peptide was submitted toN-terminal sequence analysis. Exhaustive proteolytic cleavage of thecarboxyamidomethylated peptide was performed using endoproteinase Lys-Cand endoproteinase Asp-N. Peptide fragments from these digests wereisolated on either C-3 or C-18 reversed-phase HPLC columns usingacetonitrile/water/TFA gradient elution conditions. Amino acid sequencewas determined as described in Example 4. The complete amino acidsequence determined for "cerastin" is shown in FIG. 6-1, and is comparedto that of other PAI in FIG. 12-1.

                                      TABLE 3                                     __________________________________________________________________________    Amino Acid Compositions                                                       Amino                                                                             Crotalus                                                                           Crotalus                                                                           Crotalus                                                                           Crotalus                                                                             Crotalus                                                                            Bothrops                                      acid                                                                              c. cerastes                                                                        atrox                                                                              d. durissus                                                                        d. totonatacus                                                                       h. horridus                                                                         cotiera                                       __________________________________________________________________________    Lys 3    3    3    3      4     3                                             His 0    0    0    0      0-1   0                                             Arg 5    5    5    5      4     0                                             Asx 11   10   12   12     10    10                                            Thr 5    4    4    4      3     2                                             Ser 1    2    2    2      2     1                                             Glx 7    6    5-6  5-6    6     8                                             Pro 5    6-7  4-5  5      7     6                                             Gly 9    8    10   10     8     8                                             Ala 7    6    8    8      8     9                                             Cys 12   12   12   12     10    12                                            Val 2    2    1    1      3     0                                             Met 1    0    0    0      1     0                                             Ile 0    1    1    1      0     1                                             Leu 3    3    3    3      2-3   2                                             Tyr 1    1    1    1      1     0                                             Phe 1    1    1    1      1     2                                             Trp n.d. n.d. n.d. n.d.   n.d.  n.d.                                              73-74                                                                              70-71                                                                              72-74                                                                              73-74  70-72 72                                            __________________________________________________________________________

EXAMPLE 9 Purification of PAI from Crotalus ruber ruber Venom

One gram of Crotalus ruber ruber venom (Miami Serpentarium labs, Lot#CF17SZ) was dissolved in 8 ml of 0.5M acetic acid and applied to acolumn of Sephadex G-50 fine (Pharmacia, 2.5×100cm) equilibrated at roomtemperature and eluted with 0.5M acetic acid. The column was run at aflow rate of 25 ml/hr with 5 ml fractions collected into polypropylenetubes. Aliquots of fractions were assayed for platelet aggregationinhibitory activity as described. Active fractions (61-70) were pooledand lyophilized. The dried material was resuspended in 2.0 ml of 0.5%TFA. Insoluble material was removed by centrifugation and loaded onto apreparative C-18 Water HPLC column as described and eluted employing thegradient conditions described fn Example 3. Fractions collected intopolypropylene tubes were concentrated on a Speed-Vac concentrator andanalyzed for platelet aggregation inhibitory activity.

FIG. 22 shows the activity profile for this HPLC fractionation.Individual active fractions were lyophilized. Fractions 49 and 50 werepooled and loaded onto the analytical C-18 reversed phase column andeluted using conditions described in Example 4 which consisted of anacetonitrile gradient running from 8% acetonitrile to 25% in twentyminutes followed in ten minutes to 69% acetonitrile to yield homogeneouspeptide which we have called "ruberin." Automated Edman degradation ofcarboxyamidometylated peptide give the sequence shown in FIG. 6-2.

EXAMPLE 10

Purification of PAI from Crotalus atrox

One gram of Crotalus atrox venom (Miami Serpentarium Labs, Lot #CX16AZ)was dissolved in 10 ml of 0.5M acetic acid and applied to a column ofSephadex G-50 fine (Pharmacia, 2.5×110 cm) equilibrated and run at roomtemperature with 0.5M acetic acid. The column was run at a flow rate of25 ml/hr with 5 ml fractions collected into polypropylene tubes.Aliquots of fractions were assayed for platelet aggregation inhibitoryactivity as previously described. Active fractions (81-100) were pooledand lyophilized. The dried material was dissolved in 2.0 ml of 0.5% TFAand loaded onto the preparative C-18 HPLC column and run as described inExample 3. Fractions from the column were collected into polypropylenetubes, concentrated on a Speed-vac concentrator and assayed for plateletaggregation inhibitory activity as before. Active fractions were rerunon the analytical C-18 column to yield homogeneous peptide (FIG. 23).Amino acid analysis of this material revealed that the peptide contains72 amino acids including 12 cysteine residues, as shown in Table 3. Theamino acid sequence of the isolated peptide, crotatroxin, is shown inFIG. 6-2.

EXAMPLE 11 Purification of PAI from Bothrops cotiara

Six hundred eighty milligrams of Bothrops cotiara venom (MiamiSerpentarium Labs, Lot #BO5SZ) was dissolved in 10 ml of 0.5M aceticacid and loaded onto a column of Sephadex G-50 fine (Pharmacia, 2.5×110cm) equilibrated and eluted with 0.5M acetic acid. The column was run ata flow rate of 25 ml/hr with 5 ml fractions collected into polypropylenetubes. Aliquots of fractions were assayed for platelet aggregationinhibitory activity as described previously. Active fractions (71-90)were pooled and lyophilized. Dried material was resuspended into 2.0 mlof 0.5% TFA and laded onto the waters preparative C-18 reverse-phasecolumn. The column was eluted using the conditions described in Example3. Fractions were collected into polypropylene tubes, concentrated on aSpeed-Vac concentrator and assayed for platelet aggregation inhibitoryactivity. Active fractions were individually lyophilized. Several peakfractions were rerun on the analytical C-18 column as described inExample 4. The analytical HPLC profile of homogenous peptide is shown inFIG. 24. Amino acid analysis of this material reveals this peptide tocontain 72 amino acids including 12 cysteine residues, as shown in Table3. Complete amino acid sequence of this peptide which we have called"cotiarin" is shown in FIGS. 6 and 12.

The purified peptide was tested in the receptor assays described inExample 1. Initial determinations showed that low concentrations ofcotiarin (1-4 nM) selectively inhibited vitronectin binding tovironectin receptor, whereas the same concentrations had significantlylower inhibiting activity in binding of fibrinogen to GP IIb-IIIa asshown in FIG. 25; however, subsequent experiments failed to verify thisresult.

EXAMPLE 12 Purification of PAI from Crotalus viridis lutosus

A. One gram of Crotalus viridis lutosus venom (Miami Serpentarium Labs,Lot #CL18SZ) was dissolved in 8 ml of 0.5 m acetic acid and applied to acolumn of Sephadex G-50 fine (Pharmacia, 2.5×110 cm) which wasequilibrated and eluted with 0.5M acetic acid. The column was run at aflow rate of 25 ml/hr and 5 ml fractions collected into polypropylenetubes. Aliquots of fractions were assayed for platelet aggregationinhibitory activity. Fractions (71-100) were pooled and lyophilized.Dried material was resuspended in 2.0 ml of 0.5% TFA. Insoluble materialwas removed by centrifugation and loaded onto the preparative C-18Waters reversed-phase column and eluted using the gradient elutionconditions described in Example 3. Fractions from the column werecollected into polypropylene tubes, concentrated on a Speed-Vacconcentrator and analyzed for platelet aggregation inhibitory activity.Active fractions were lyophilized in their individual tubes. Fractionswith peak activity were rerun on the Waters analytical C-18 column usingthe acetonitrile gradient described in Example 4. Fractions werecollected by hand into 1.5 ml Eppendorf tubes. Homogeneous fractionswere pooled and lyophilized. Analytical HPLC of this material showed asingle symmetric peak. The complete amino acid sequence of this peptidewhich we have called "lutosin" is shown in FIG. 6-3 and FIG. 12-1.

B. In a similar manner to that set forth in paragraph A, the PAIs fromB. jararacussu, C. basificus, C. durissus durissus, C. v. oreganus, C.h. horriddus, C. v. helferi, C. durissus totonactus and from C. m.molossus, were isolated and purified. Amino acid compositions forseveral of these peptides are shown in Table 3. The amino acid sequencesof the PAI from C. h. horridus, C. basilicus, and C. m. molossus,designated horridin, basilicin and molossin, respectively, are shown inFIG. 6. Receptor binding data for the purified peptides of Examples 1-12are shown in FIG. 26.

In Examples 13-16 below, peptides were synthesized by solid-phasetechniques on an Applied Biosystems 431A Peptide Synthesizer using t-Bocamino acids activated as HOBt active esters in accordance with theinstructions of the manufacturer, which are briefly as follows for thepreparation of Boc-AA1 . . . AA(n-1)-AA(n)-O-PAM-polystyrene resin.

One-half mmol of selected Boc-AA(n)-O-PAM-polystyrene resin is treatedaccording to the following schedule for incorporation of theBoc-AA(n-1)-OH:

1) TFA deprotection: 30% TFA in DCM, 3 min, 50% TFA in DCM, 16 min.

2) Washes and neutralizations: DCM washes (5×), 3 min, 5% DIEA in DCM, 2min, 5% DIEA in NMP, 2 min. NMP wash (6×), 5 min.

3) Coupling: 4 equivalents Boc-AA-HOBt ester in NMP (preactivate 55min), 38 min, DMSO to make 15% DMSO/85% NMP, 16 min, 3.8 equiv DIEA, 5min.

4) Wash and resin sample: NMP wash, 3 min.

5) Capping: 10% acetic anhydride, 5% DIEA in NMP, 8 min.

6) Washes: DCM washes (6×) 4 min.

EXAMPLE 13 Preparation of Analog #1 E²⁸ L⁴¹ C⁶⁴ !barbourin (28-73):E-C-A-D-G-L-C-C-D-Q-C-R-F-L-K-K-G-T-V-C-R-V-A-K-G-D-W-N-D-D-T-C-T-G-Q-S-C-D-C-P-R-N-G-L-Y-G

One-half mmol of PAM-Gly resin (0.6 meq/g, Applied Biosystems, FosterCity, Calif.) was subjected to Procedure A with the required amino acids(introduced in order). The Boc-protected amino acids had the followingside-chain protection: Arg(Tos), Asp(OcHex), Cys(4-MeBzl), Glu(OcHex),Lys (Cl-Z), Thr(OBzl), Trp(CHO), and Tyr(Br-Z). Following assembly ofthe completed protected peptide-resin chain, the amino terminalBoc-group was removed with TFA and the resin dried as its TFA-salt form.The resin (1.3 g) was subjected to "low-high" HF deprotection protocolsfollowed by removal of HF "in vacuo". The dried peptide-resin mixturewas transferred to a fritted funnel (coarse) with ethyl ether and waswashed several times with alternate washes of ether and chloroform toremove most of the organic protecting groups and scavengers used in thedeprotection.

The peptide mixture was transferred to 2 L of 0.4% acetic acid and thepH adjusted to 7.99 with concentrated NH₄ OH. The resin was filteredfrom this solution and the solution allowed to sit at 4° C. withoutstirring for 20 hr. This was followed by warming the solution to roomtemperature and storing for 3 days again without stirring. Precipitatedmaterial was removed by filtration and the supernatant pH adjusted to3.0 with acetic acid and lyophilized.

The crude material was dissolved in 8.0 ml of 0.5M acetic acid andloaded onto a Sephadex G-50 fine column (2.5×100 cm) equilibrated with0.5M acetic acid. The column was run at 20 ml/hr and fractions (4 ml)were collected into polypropylene tubes. Aliquots of fractions weredried, resuspended in water and tested for platelet aggregationinhibitory activity as previously described. Active fractions (71-90)were pooled and lyophilized.

Dried material (66 mg) was redissolved in 2.0 ml of 0.1M acetic acid andloaded onto the Waters Preparative C-18 column equilibrated with 8%acetonitrile containing 0.1% TFA. A gradient running from 8%acetonitrile to 20% in 10 minutes followed by a slow gradient to 30%acetonitrile in 40 min was performed. The column was eluted at 18 ml/minand fractions (12 sec) were collected into polypropylene tubes.Fractions were concentrated on a Speed-Vac concentrator to 1.0 ml volumeand 10 ul aliquots were tested in the platelet aggregation assay.

Active fractions (29-32) were individually lyophilized and analyzed onthe analytical C-18 HPLC column with an 8-30% acetonitrile gradient.Fractions 29 and 30 were pooled and loaded onto the analytical column in1.0 ml of 0.5% TFA. The major peak was collected manually andlyophilized to yield 1.6 mg of pure peptide.

Amino acid analysis of this material confirmed the identity of thepeptide. Assay of this material for its ability to inhibit the bindingof fibrinogen to GP IIb-IIIa and vitronectin to VnR is displayed inFIGS. 26 and 27. These data demonstrate the high affinity of this analogfor GP IIb-IIIa and the relative lack of affinity for VnR atconcentrations up to 1 uM.

EXAMPLE 14 Preparation of Analog #2, K²⁹ !eristicophin (4-51):E-w-P-C-A-T-G-P-C-C-R-R-C-K-F-K-R-A-G-K-V-C-R-V-A-K-G-D-W-N-N-D-Y-C-T-G-K-S-C-D-C-P-R-N-P-W-N-G

One-half mmol of PAM-Gly resin (0.6 meq/g, Applied Biosystems, FosterCity, Calif.) was subjected to Procedure A with the required amino acids(introduced in order). The Boc-protected amino acids had the followingside-chain protection: Arg(Tos), Asp(OcHex), Cys(4-MeBzl), Glu(O-cHex),Lys(Cl-Z), Ser(OBzl), Thr(OBzl), Trp(CHO) and Tyr(Br-Z). Cleavage,refolding and purification of this peptide was identical to the previousexamples. Receptor binding data for this analog are shown in FIGS. 26and 28.

EXAMPLE 15 Preparation of Analog #3: G-C-G-K-G-D-W-P-C-A-NH₂

One-half mmol of pMBHA resin (0.72 meq/g, Applied Biosystems, FosterCity, Calif.) was subjected to Procedure A with the required amino acids(introduced in order). The Boc-protected amino acids had the followingside-chain protection: Asp(O-cHex), Cys(4-MeBzl), and Lys(Cl-Z).Following completion of the assembly of the protected peptide-resin, theamino terminal Boc group was removed with TFA and the resin dried as itsTFA-salt form. The resin (1.54 g) was treated with anhydrous hydrogenfluoride (HF) containing 10% anisole, 2% ethyl methyl sulfide for 30 minat -10° C., and an additional 30 min at 0° C. The HF was removed invacuo and the peptide/resin mixture was suspended in diethyl etherfollowed by alternately washing with chloroform and ether 3×. After afinal ether wash, the peptide was extracted from the resin with 2.0Macetic acid, diluted with distilled water and lyophilized.

The crude peptide (370 mg) was dissolved in deoxygenated 10 mM NH₄ OAc,pH 8, to 0.5 mg/ml and allowed to oxidize by dropwise addition of aslight excess of 0.01M potassium ferricyanide (K₃ Fe(CN)₆) solution,stirred an additional 20 min, and adjusted to pH 5 with acetic acid. Thepeptide solution was treated with DOWEX AG3×4 anion-exchange resin for15 min with stirring and the resin filtered, diluted with H₂ O andlyophilized to yield the crude cyclized peptide. The crude cyclizedpeptide (392 mg) was purified by despairing on Sephadex G-25F using 0.5Macetic acid as eluent, followed by ion-exchange chromatography onCM-Sepharose (Pharmacia) using an elution gradient generated by additionof 100 mM NH₄ OAc to a solution of 10 mM NH₄ OAc, pH 4.5. Fractionswhich had a minimum purity of 90% by HPLC analysis were pooled andlyophilized from H₂ O several time to yield 175 mg. Final purificationconsisted of preparative HPLC purification on a Water C-18 reverse-phasecolumn with an acetonitrile/water/TFA gradient to yield purifiedpeptide. Receptor binding data for this analog are shown in FIGS. 26,29B and 30.

EXAMPLE 16 Preparation of Additional Analogs

The following analogs were synthesized; in most cases in a mannersimilar to that set forth in Example 15. However, analog 60, shownbelow, was prepared in solution via guanidation of the side chain of thelysine residue of analog #19 using the procedure of Bajusz, S., et al.,FEBS Letts (1980) 110:85-87.

One mg of analog #19 was reacted with 1 mg of1-amidino-3,5-dimethylpyrazole nitrate (Aldrich) in 1 ml of absoluteethanol in the presence of diisopropylethylamine (DIEA) at roomtemperature for 4 days. The product analog 60 was purified from excessreagent and starting materials by reversed-phase HPLC on a C-18 columnusing a gradient of acetonitrile in 0.1% trifluoroacetic acid. Ninehundred ug of this material was isolated in purified form.

#4 GC-K-G-D-W-P-C-A-NH₂

#5 C-G-K-G-D-W-P-C-NH₂

#6 G-C-G-K-G-D-W-C-A-NH₂

#7 G-C-K-G-D-W-C-A-NH₂

#8 AcetyI-C-K-G-D-C-NH₂

#9Mpr-K-G-D-Pen-NH₂

#10 C-K-G-D-W-P-C-NH₂

#11 Acetyl-C-R-G-D-Pen-NH₂

#12 C-K-G-D-Y-P-C-NH₂

#13 C-K-G-D-F-P-C-NH₂

#19Mpr-K-G-D-W-P-C-NH₂

#34 C-K-G-D-W-G-C-NH₂

#35 C-K-G-E-W-P-C-NH₂

#36 C-Orn-G-D-W-P-C-NH₂

#37: C-K-A-D-W-P-C-NH₂

#38: C-K-A.sup.↑ -D-W-P-C-NH₂

#39: C-K-G-D-W-(Sar)-C-NH₂

#40: C-K(Formyl)-G-D-W-P-C-NH₂

#41: C-K-G-D-I-P-C-NH₂

#42: C-K-G-D-(4-Cl-Phe)-P-NH₂

#43: C-K-(Sar)-D-W-P-C-NH₂

#44: C-K-G-D-(4-NO₂ -Phe )-P-C-NH₂

#45: C-K-G-D-(NMePhe)-P-C-NH₂

#46: C-H-G-D-W-P-C-NH₂

#47: Acetyl-C-K-G-D-W-P-C-NH₂

#48: Mpr-K-G-D-W(Formyl)-P-C-NH₂

#49: Mvl-K-G-D-W-P-C-NH₂

#50: MprK-G-D-W.sup.↑ -P-Pen-NH₂

#51: Mpr-K-G-D-W-P-Pen-NH₂

#52: MprK-G-D-W-P-Pen.sup.↑ -NH₂

#53: MprK-G-D-W-P.sup.↑ -Pen-NH₂

#54: MprK-G-D.sup.↑ -W-P-Pen-NH₂

#55: Mpr-K-G-D-W-(Thz)-C-NH₂

#56: Mpr-K-G-D-H(2,4-DNP)-P-C-NH₂

#57: Mpr-K-G-D-(2-Nal)-P-Pen-NH₂

#58: Mvl-K-G-D-W-P-Pen-NH₂

#59: Mpr-K-G-D-W-(PiP)-Pen-NH₂

#60: Mpr-(Har)-G-D-W-P-C-NH₂

#61: Mpr-K-G-D-W-P-C.sup.↑ -NH₂

#62: Mpr-(D-Lys)-G-D-W-P-Pen-NH₂

#63: Mpr-(Har)-G-D-W-P-Pen-NH₂

#64: Mpr-(Acetimidyl-LYs)-G-D-W-P-C-NH₂

#65: Mpr-(Acetimidyl-LYs)-G-D-W-P-Pen-NH₂

#66: Mpr-(N^(G),N^(G') -ethylene-Har)-G-D-W-P-C-NH₂

#67: Mpr-(N^(G),N^(G') -ethylene-Har)-G-D-W-P-Pen-NH₂

#68: Mpr-Har-Sar-D-W-P-C-NH₂

#69: Mpr-(Acetimidyl-LYs)-G-D-W-P-Pen-NH₂

EXAMPLE 17 PAI Activity of Peptides

When tested in the standard aggregation inhibition assays describedabove, analogs #3-5 had IC₅₀ values of 5 uM for ability to inhibitADP-induced human platelet aggregation. However, analog #6 has an IC₅₀of more than 200 uM, and analog #7, 100 uM. IC₅₀ values for the analogsof the invention in this assay are as follows:

    ______________________________________                                                                        Appr.                                         Analog                                                                              Sequence                  IC.sub.50 (uM)                                ______________________________________                                        #3    G--C--G--K--G--D--W--P--C--A--NH.sub.2                                                                  5                                             #4    G--C--K--G--D--W--P--C--A--NH.sub.2                                                                     5                                             #5    C--G--K--G--D--W--P--C--NH.sub.2                                                                        5                                             #6    G--C--G--K--G--D--W--C--A--NH.sub.2                                                                     >200                                          #7    G--C--K--G--D--W--C--A--NH.sub.2                                                                        100                                           #8    Acetyl-C--K--G--D--C--NH.sub.2                                                                          200                                           #9    Mpr--K--G--D--Pen--NH.sub.2                                                                             25                                            #10   C--K--G--D--W--P--C--NH.sub.2                                                                           5                                             #11   Acetyl-C--R--G--D--Pen--NH.sub.2                                                                        5                                             #12   C--K--G--D--Y--P--C--NH.sub.2                                                                           12                                            #13   C--K--G--D--F--P--C--NH.sub.2                                                                           20                                            #19   Mpr--K--G--D--W--P--C--NH.sub.2                                                                         1                                             #34   C--K--G--D--W--G--C--NH.sub.2                                                                           100                                           #35   C--K--G--E--W--P--C--NH.sub.2                                                                           >300                                          #36   C--Orn--G--D--W--P--C--NH.sub.2                                                                         150-200                                       #37   C--K--A--D--W--P--C--NH.sub.2                                                                           100                                           #38   C--K--A.sup.↑ --D--W--P--C--NH.sub.2                                                              >200                                          #39   C--K--G--D--W--(Sar)--C--NH.sub.2                                                                       5                                             #40   C--K(Formyl)--G--D--W--P--C--NH.sub.2                                                                   >200                                          #41   C--K--G--D--I--P--C--NH.sub.2                                                                           100                                           #42   C--K--G--D--(4-Cl--Phe)--P--NH.sub.2                                                                    20                                            #43   C--K--(Sar)--D--W--P--C--NH.sub.2                                                                       50                                            #44   C--K--G--D--(4-NO.sub.2 --Phe)--P--C--NH.sub.2                                                          75                                            #45   C--K--G--D--(NMePhe)--P--C--NH.sub.2                                                                    >200                                          #46   C--H--G--D--W--P--C--NH.sub.2                                                                           200                                           #47   Acetyl-C--K--G--D--W--P--C--NH.sub.2                                                                    2.5                                           #48   Mpr--K--G--D--W(Formyl)--P--C--NH.sub.2                                                                 1                                             #49   Mvl--K--G--D--W--P--C--NH.sub.2                                                                         1.5                                           #50   Mpr--K--G--D--W.sup.↑ --P--Pen--NH.sub.2                                                          >200                                          #51   Mpr--K--G--D--W--P--Pen--NH.sub.2                                                                       0.75                                          #52   Mpr--K--G--D--W--P--Pen.sup.↑ --NH.sub.2                                                          5                                             #53   Mpr--K--G--D--W--P.sup.↑ --Pen--NH.sub.2                                                          >200                                          #54   Mpr--K--G--D.sup.↑ --W--P--Pen--NH.sub.2                                                          >100                                          #55   Mpr--K--G--D--W--(Thz)--C--NH.sub.2                                                                     2                                             #56   Mpr--K--G--D--H(2,4-DNP)--P--C--NH.sub.2                                                                5                                             #57   Mpr--K--G--D-(2-Nal)--P--Pen--NH.sub.2                                                                  1                                             #58   Mvl--K--G--D--W--P--Pen--NH.sub.2                                                                       1                                             #59   Mpr--K--G--D--W--(Pip)--Pen--NH.sub.2                                                                   1                                             #60   Mpr--(Har)--G--D--W--P--C--NH.sub.2                                                                     0.15                                          #61   Mpr--K--G--D--W--P--C.sup.↑ --NH.sub.2                                                            15                                            #62   Mpr--K.sup.↑ --G--D--W--P--Pen--NH.sub.2                                                          2.5                                           #63   Mpr--(Har)--G--D--W--P--Pen--NH.sub.2                                                                   0.10                                          #64   Mpr--(Acetimidyl-Lys)--G--D--W--P--C--NH.sub.2                                                          0.25                                          #68   Mpr--Har--Sar--D--W--P--C--NH.sub.2                                                                     3.0                                           #69   Mpr--(Acetimidyl-         0.5                                                 Lys)--G--D--W--P--Pen--NH.sub.2                                         ______________________________________                                    

EXAMPLE 18 Activity of Linear versus Cyclic Peptides

When tested for inhibition of fibrinogen binding to GP IIb-IIIa in theplate assay, linear RGDW-NH₂ was very similar in activity to cyclicGCGRGDWPCA-NH₂ (FIG. 29A). In contrast, the linear KGDW-NH₂ was muchless potent than cyclic GCGKGDWPCA-NH₂ (FIG. 29B). For the KGDWcompounds, but not the RGDW compounds, cyclization resulted in a markedincrease in the ability of the peptide to inhibit the binding offibrinogen to GP IIb-IIIa.

EXAMPLE 19 Results of Plate Binding Assays for Synthetic Peptides

The peptides synthesized in Example 17, in addition to being assessedfor the ability to inhibit platelet aggregation directly, were alsotested in the plate assays of the invention as described above. Theresults for analogs 4, 5, 6, 7, and 8 are shown in FIGS. 30C, 30D, 30E,30A, and 30B, respectively. As indicated in the figure, these analogsare differentially capable, to varying degrees, of inhibiting thebinding of fibrinogen to GP IIb-IIIa as compared to vitronectin tovitronectin receptor. Analog #4 appears, among this group, to have thehighest differential. Analogs #7 and #5, on the other hand, are alsoquite specific, and have excellent platelet aggregation inhibitionactivities.

EXAMPLE 20 Effects of Purified Peptides on Cell Adhesion

M21 melanoma cells were labelled with ³⁵ S-methionine, and then added tovitronectin-coated plates in the presence of the indicatedconcentrations of purified snake venom peptides. Cell attachment wasmeasured by solubilizing the cells remaining after an incubation andwash, as described in Section C, on page 40. As shown in FIG. 31,neither barbourin nor Peptide 1 (truncated barbourin) had a significanteffect on cell adhesion to vitronectin, although both are potentinhibitors of platelet aggregation as shown in Examples 2 and 3. Incontrast, cotiarin, which is a potent inhibitor of vitronectin bindingto the vitronectin receptor, was very potent in inhibiting cellattachment to vitronectin. In similar experiment, Peptide #3, Peptide #3with K replaced by R (GCGRGDWPCA-NH₂) and RGDS were examined on M21 cellattachment to vitronectin. As shown in FIG. 32, RGDS and GCGRGDWPCA-NH₂are potent inhibitors of cell attachment whereas GCGKGDWPCA-NH₂ wasineffective up to 60 uM.

EXAMPLE 21 Comparison of Analogs 60 and 19

Analogs 60 and 19 described above are peptides of the inventioncontaining the sequence K*GDX and are identical except for theembodiment of K* . Analog 60 is of the formula:

Mpr-(Har)-G-D-W-P-C-NH₂ ;

analog 19 is of the formula:

Mpr-K-G-D-W-P-C-NH₂.

These analogs were tested by standard platelet aggregation inhibitionassays and using the cell adhesion assay of Example 20 above. Theresults are shown in FIGS. 33 and 34. As shown in FIG. 33, analog #60 isefficient at vanishingly small concentrations in inhibiting plateletaggregation, and is relatively less effective in preventing celladhesion to victronectin. FIG. 34 shows analog #19 has good plateletaggregation inhibition activity as well as specificity; however, it isless active in the platelet aggregation inhibition assay than its analog#60 counterpart. Analog #60 has an IC₅₀ in platelet aggregation ofapproximately 0.15 nM; analog #19 has an IC₅₀ of approximately 1 nM.

EXAMPLE 22 Folt's Model of Thrombosis in Dog Coronary Artery

A. Initiation of cyclic flow reductions (CFRs) in open chest dog. Anoccluder placed on the left anterior descending (LAD) coronary artery ofa 20 kg dog, as previously described was performed. The phasic and meanblood flows as measured by an elecromagnetic (EM) flow probe, andDoppler flow probe are shown in FIG. 35.

B. Effect of Cyclic GCGKGDWPCA-NH2 (Analog #3) on the CFRs in the openchest dog. A dose of 10 mg of this peptide was infused into a peripheralvein in the dog. Shown in FIG. 36 are the blood flow patterns in theLAD, as described above. Note the partial ablation of the CFRs, as seenin the decreased slope of the flow reductions. Note also that flow isnot reduced to the same degree as in the control (A).

C. A second infusion of 40 mg of Analog #3 was given into a peripheralvein. As shown in FIG. 37 the complete ablation of the CFRs indicatesthat full flow has been restored in the LAD.

EXAMPLE 23 Construction of Expression Vectors for Barbourin Peptides

A gene encoding the full length L⁴¹ ! barbourin peptide (1-73) wasassembled from synthetic oligonucleotides as shown in FIG. 38, whichwere kinased, annealed and ligated into EcoRI-HindIII digested M13mp18using standard procedures. The bacterial alkaline phosphatase gene(phoA) signal sequence (Watson, M. E. E., Nucleic Acids Research (1984)12:5145) was added to the barbourin construct by ligating syntheticoligonucleotides into the EcoRI/NcoI sites of the L⁴¹ ! barbourin (1-73)construct as shown in FIG. 39. The nucleotide sequences of allconstructs were verified by the Sanger dideoxy chain termination method.

A truncated version of this peptide was also constructed from syntheticoligonucleotides which would encode only amino acids 28-73 of the fulllength molecule. Two alterations, Q²⁸ to E²⁸ and A⁶⁴ to C⁶⁴ wereintroduced using site directed mutagenesis as described by Kunkel et al.Meth Enzymol (1987) 154:367. The phoA signal sequence was added to thetruncated version as described above (FIG. 40). In addition, the signalsequence for the E. coli heat-stable enterotoxin II (Picken, R. W., etal. Infect Immun (1983) 42:269) was added to the truncated version usingsynthetic oligonucleotides with EcoRI and NcoI compatible ends. Allbacterial secretion constructs were subcloned into the bacterialexpression vector pPROK-1 (Brosius, J., Gene (1984) 27:151, ibid:161),abailable commercially from CLONTECH Lab, Inc. using EcoRI and HindIIIrestriction endonucleases.

A gene encoding tandem repeats of the desired title peptide was preparedusing the polymerase chain reaction (PCR) to produce the multimerizationunit from the full-length barbourin peptide 1-73 containing L41 and C64.

FIGS. 41A and 41B shows the oligonucleotides used for the PCR synthesis.The PCR reaction was conducted according to the method of Saiki, R. K.,et al. Science (1988) 239:487. The resulting polymer junction containsmethionines at either end of the sequence as shown in FIG. 42 andprovides desirable restriction sites for the construct.

The tandem repeats are formed from the individual multimer-formingcomponents by, for example, ligating an EcoRI/BamHI fragment to aBglII/HindIII fragment in an M13mp18 vector cut with EcoRI/HindIII toform a dimer. The resultant dimer is excised with EcoRI and BamHI andreligated to a BglII/HindIII fragment to produce a trimer, and so onuntil the desired size is obtained. This construction is diagramed inFIGS. 43A and 43B.

The multimer was then ligated into the E. coli vector pKK233-2, Amann,E., et al., Gene (1985) 40:183, available from Clontech, by digestingthe vector with NcoI/HindIII and ligating a monomer subfragment ofNcoI/BamHI and multimer subfragments of BglII/HindIII.

For expression as a fusion protein, the above digested vector was usedalong with an NcoI-EcoRI subfragment containing a slightly modifiedamino-terminal portion (amino acids 1 to 72) of the chloramphenicolacetyltransferase gene (Chang, C. N., et al. Gene (1987) 55:189) andEcoRI-HindIII subfragments of the multimer constructions.

EXAMPLE 24 Expression of Recombinant Genes

Protein expression from all of the recombinant plasmids described aboveis induced according to Kanamari et al. Gene (1988) 66:295 aftertransfection into appropriate E. coli host strains. Products arecharacterized by sodium dodecyl sulfate polyacrylamide gelelectrophoresis and by their ability to inhibit ADP-induced plateletaggregation in platelet-rich plasma. Following purification, themultimeric proteins are converted to monomer units with cyanogen bromidecleavage and the products assayed as above.

We claim:
 1. A method to determine the presence or absence of plateletaggregation inhibitor (PAI) activity in a biological fluid, which methodcomprises:contacting a sample of said fluid with a solid support coatedwith purified platelet GP IIb-IIIa in the presence of a solution offibrinogen (Fg) or von Willebrand Factor (vWF) under conditions whereinFg or vWF binds to said GP IIb-IIIa, and detecting the decrease or lackof decrease in the binding of Fg or vWF to GP IIb-IIIa in comparison tothe binding of Fg or vWF to GP IIb-IIIa in a control which does notcontain biological fluid.